Culture material and its use

Abstract

[Problem] To provide a culture member and a culture instrument by which a spheroid can be formed by culturing cells, etc. [Solution] A culture member for culturing cells, a tissue or an organ on a culture surface thereof, said culture member comprising a 4-methyl-1-pentene polymer (X), having a water contact angle of the culture surface of more than 100° and not more than 160°, and having an oxygen permeability at a temperature of 23°C and humidity of 0% of 4500 to 90000 cm3 / (m2 × 24 h × atm).

Description

[Technical Field]

[0001] The present invention relates to a culture device and its use. [Background technology]

[0002] In living organisms, cells form three-dimensional networks to express their functions. Therefore, in order to fully utilize the intrinsic functions of cells, spheroids, which are cultured in three dimensions rather than conventional planar cell cultures, have been attracting attention. Spheroids have been reported to produce results that are closer to the living body than planar cell cultures in cell function evaluation and drug screening. Spheroids are expected to be an important tool for bridging the gap between in vitro and in vivo testing in drug discovery. Stem cells, including iPS cells, are expected to be an important cell source for regenerative medicine and cell therapy, and the preparation of large quantities of high-quality cells is required. 3D culture of stem cells to form spheroids allows for high-density culture while maintaining the cells' inherent pluripotency (undifferentiated state), and spheroids are attracting attention for their potential utility in regenerative medicine. Accordingly, there is a growing demand for new cell culture technologies that can produce large quantities of uniformly shaped and sized cell spheroids in a stable and easy manner.

[0003] Given the above circumstances, various methods for forming spheroids have been developed. These include, for example, a method using a culture vessel whose surface has been processed or treated to inhibit cell adhesion (Patent Document 1), a method in which cells are cultured in a suspended state in a vessel formed with a resin layer with low cell adhesiveness (Patent Document 2), and a method in which spheroids are formed in an attached state on the surface of the culture vessel by increasing the amount of proteoglycan adsorption of the culture vessel to a specific value or more (Patent Document 3). In particular, when culturing non-adhesive cells to form spheroids, the surface of the culture vessel is usually treated to prevent cells from adhering (for example, by subjecting the surface of the culture vessel to a superhydrophilic treatment, rendering it hydrophobic, or giving it a structure that makes it less likely to adhere). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020 / 013345 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-061609 [Patent Document 3] Japanese Patent Application Laid-Open No. 2017-77241 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention was made in consideration of the above circumstances, and aims to provide a culture member and culture instrument capable of culturing cells and the like to form spheroids. Another aim is to provide a culture member and culture instrument that has excellent shape stability, can create an oxygen environment suitable for culturing cells, tissues, or organs, does not emit autofluorescence, does not impair cell observation, and is resistant to drug sorption. A further aim is to provide a culture member and culture instrument that can culture differentiated cells, particularly stem cell-derived cells, with high differentiated cell purity. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that the above problems can be solved by a culture device having the following configuration, and have completed the present invention. The present invention relates to, for example, the following [1] to

[12] . [1] A culture member for culturing cells, tissues, or organs on its culture surface, the culture member comprising a 4-methyl-1-pentene polymer (X), the water contact angle of the culture surface being greater than 100° and not greater than 160°, and the oxygen permeability at a temperature of 23°C and humidity of 0% being 4500 to 90000 cm 3 / (m 2 × 24h × atm). [2] The culture element according to [1], wherein the 4-methyl-1-pentene polymer (X) is a copolymer (x1) of 4-methyl-1-pentene with at least one selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). [3] The culture member according to [1] or [2], wherein the culture surface is not subjected to processing to form an uneven structure. [4] The culture member according to any one of [1] to [3], which is for forming spheroids. [5] The culture member according to any one of [1] to [4], wherein the cells, tissues, or organs include differentiated cells derived from iPS cells. [6] The culture member according to any one of [1] to [5], wherein the cells, tissues, or organs include cardiomyocytes. [7] The culture member according to any one of [1] to [6], wherein the culture member is in the form of a film or a sheet. [8] A culture instrument, at least the culture surface of which is formed from the culture member according to any one of [1] to [7]. [9] A step (A) of contacting a cell, tissue, or organ with the culture surface of the culture member according to any one of [1] to [7] or the culture instrument according to [8]; and A method for culturing cells, tissues, or organs, comprising the step (B) of culturing the cells, tissues, or organs in contact with the culture surface to form spheroids.

[10] The method for culturing cells, tissues, or organs according to [9], further comprising a step (C) of inducing differentiation of the cells, etc., wherein step (C) is a step of inducing differentiation of iPS cells into cardiomyocytes by protein-free cardiac differentiation induction (PFCD).

[11] The method for culturing cells, tissues, or organs according to [9] or

[10] , which increases myocardial purity.

[12] A spheroid formed by the culture method according to any one of [9] to

[11] . [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a culture element and a culture instrument that can culture cells and the like to form spheroids. Furthermore, according to the present invention, it is possible to provide a culture element and a culture instrument that have excellent shape stability, can realize an oxygen environment suitable for culturing cells, tissues, or organs, does not emit autofluorescence, does not impair cell observation, and is resistant to drug sorption. Furthermore, according to the present invention, it is possible to provide a culture element and a culture instrument that can culture differentiated cells, particularly stem cell-derived cells, with high differentiated cell purity. Furthermore, the culture element and the culture instrument of the present invention facilitate the differentiation of iPS cells into cardiomyocytes. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the schedule of an experiment for inducing differentiation of iPS cells into cardiomyocytes. [Figure 2] FIG. 2 shows photographs of the morphology of cells on day 12 of induction of cardiac differentiation. [Figure 3] FIG. 3 shows photographs of the morphology of cells on day 19 of induction of cardiac differentiation. [Figure 4] FIG. 4 is a photograph showing the spheroid formation of human osteosarcoma-derived cancer cells (HOF-143B) in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0009] Unless otherwise specified, the expression "A to B" regarding a numerical range means that the range is from A to B. For example, the expression "1 to 5%" means from 1% to 5%.

[0010] [Culture materials] The culture member according to the present invention is a member for culturing cells, tissues, or organs (hereinafter also referred to as cells, etc.) on its culture surface, and the culture member contains a 4-methyl-1-pentene polymer (X), and the culture surface has a water contact angle of more than 100° and not more than 160°, and an oxygen permeability of 4500 to 90000 cm at a temperature of 23°C and a humidity of 0%. 3 / (m 2×24h×atm). Here, the term "culture component" refers to a component that constitutes at least a part of a culture instrument used for culturing cells, etc. When the culture component is part of the culture instrument, at least the culture surface on which cells, etc. are cultured is constituted by the culture component of the present invention. Here, the term "culture surface" refers to a surface on which a culture medium is formed, a surface on which cells, etc. are seeded, or a surface on which a culture medium is formed and cells, etc. are seeded, when culturing cells, etc. In other words, the term "culture surface" is a concept that encompasses both a surface on which a culture medium is to be formed and a surface on which cells, etc. are to be seeded.

[0011] In this specification, the term "culture" is used in a broad sense to include not only the proliferation and maintenance of cells, etc., but also processes such as seeding, subculturing, differentiation induction, and self-organization induction of cells, etc.

[0012] The form of the culture member of the present invention is not particularly limited, and may be, for example, a film or sheet. When the culture member is in a film or sheet form, at least one surface of the film or sheet-like culture member can be suitably used as a culture device with the culture surface.

[0013] The culture member of the present invention means a member whose culture surface is not coated with a natural polymer material, synthetic polymer material, or inorganic material that serves as a scaffold for cells or the like.

[0014] The thickness of the culture member of the present invention is not particularly limited. Furthermore, the thickness of the culture member of the present invention is not particularly limited, but is preferably 20 to 500 μm, more preferably 25 to 400 μm, and particularly preferably 50 to 200 μm. When the thickness of the culture member is within the above range, an appropriate oxygen concentration in the medium necessary for cell proliferation can be obtained, and a suitable culture tool can be easily produced without bending of the culture member, particularly the bottom surface of the culture vessel.

[0015] When the culture device of the present invention is placed on the bottom of a container to prepare a culture device such as a dish (also called a petri dish), flask, insert, or plate, the thickness of the culture device is not particularly limited, but is preferably 20 μm to 400 μm, more preferably 20 μm to 300 μm, and even more preferably 20 μm to 200 μm. The thickness of the culture device is selected appropriately depending on the form of the culture device, but by adjusting the thickness within the above range, it becomes easier to obtain an appropriate oxygen concentration in the medium necessary for cell proliferation, and it becomes easier to prepare a suitable culture device with sufficient strength.

[0016] The surface of the culture member of the present invention may be processed as long as the effects of the present invention are not impaired. Examples of surface processing include surface modification treatments such as forming a concave-convex structure, hydrophilization treatment, and hydrophobic treatment. The culture member of the present invention can form spheroids because cells do not easily adhere to it even without processing its surface. Therefore, it is preferable that the surface of the culture member of the present invention is not processed, and it is more preferable that the surface is not processed to form an uneven structure.

[0017] The method used for surface modification is not particularly limited, but examples include hydrophilization treatments such as corona treatment, plasma treatment, ozone treatment, and ultraviolet treatment; hydrophobic treatments such as esterification, silylation, and fluorination; surface graft polymerization, chemical vapor deposition, etching; addition of specific functional groups such as hydroxyl groups, amino groups, sulfonic groups, thiol groups, and carboxyl groups; treatments using specific functional groups such as silane coupling, titanium coupling, and zirconium coupling; surface roughening using oxidizing agents; and physical treatments such as rubbing and sandblasting. These surface modification treatments may be performed alone or in combination of two or more. When performing surface modification treatments, it is preferable to perform them at least on the culture surface.

[0018] The method for producing the culture device of the present invention is not particularly limited, and the equipment used for production is also not limited. For example, a film or sheet containing the 4-methyl-1-pentene polymer (X) can be formed, and the film or sheet can be molded into a desired shape as needed to produce the culture device. Films, sheets, or other molded products that can be used as culture devices can also be obtained by direct molding using methods such as extrusion molding, solution casting, injection molding, and blow molding.

[0019] Specific examples of methods for forming the film or sheet include the usual inflation method and T-die extrusion method. The production is usually carried out under heating. When the T-die extrusion method is used, the extrusion temperature is preferably 100°C to 400°C, and more preferably 200°C to 300°C. The roll temperature is preferably 45°C to 75°C, and more preferably 55°C to 65°C.

[0020] Alternatively, the film or sheet may be produced by a solution casting method in which the 4-methyl-1-pentene polymer (X) is dissolved in a solvent, poured onto a resin or metal, and slowly dried while leveling to form a film (sheet). There are no particular limitations on the solvent used, and hydrocarbon solvents such as cyclohexane, hexane, decane, and toluene may be used. Two or more solvents may be mixed, taking into consideration the solubility of the 4-methyl-1-pentene polymer (X) and drying efficiency. The polymer solution can be applied by a method such as table coating, spin coating, dip coating, die coating, spray coating, bar coating, roll coating, or curtain flow coating, followed by drying and peeling to form a film or sheet.

[0021] The culture device of the present invention is preferably a culture device for spheroid formation, more preferably a cell culture device for spheroid formation.

[0022] [4-methyl-1-pentene polymer] In the present invention, 4-methyl-1-pentene homopolymers and copolymers of 4-methyl-1-pentene with other monomers are collectively referred to as "4-methyl-1-pentene polymer (X)". A copolymer of 4-methyl-1-pentene and another monomer, which is an example of a 4-methyl-1-pentene polymer, may be any of a random copolymer, an alternating copolymer, a block copolymer, and a graft copolymer. As a copolymer of 4-methyl-1-pentene and another monomer, a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is preferred because it has high strength and is resistant to tearing, cracking, and bending even when used as a member.

[0023] The 4-methyl-1-pentene polymer is preferably at least one polymer selected from a 4-methyl-1-pentene homopolymer and a copolymer of 4-methyl-1-pentene with at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), and more preferably a copolymer of 4-methyl-1-pentene with at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0024] Examples of the olefin include ethylene, propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and 1-eicosene. The olefin can be appropriately selected depending on the physical properties required for the culture medium. For example, from the viewpoints of appropriate oxygen permeability and excellent rigidity, the olefin is preferably an α-olefin having 8 to 18 carbon atoms, and more preferably at least one selected from 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, and 1-octadecene. When the carbon number of the olefin is within the above range, the polymer has better processability and tends to be less susceptible to poor appearance of the culture medium due to cracks or edge breakage. Furthermore, the rate of defective culture mediums is reduced.

[0025] The olefin may be one or more types. From the viewpoint of material strength, the carbon number is preferably 2 or more, and more preferably 10 or more. When combining two or more different α-olefins, it is particularly preferable to combine at least one selected from 1-tetradecene and 1-hexadecene with at least one selected from 1-heptadecene and 1-octadecene.

[0026] The content of structural units derived from 4-methyl-1-pentene in the 4-methyl-1-pentene polymer is preferably 60 to 100 mol %, more preferably 80 to 98 mol %. Furthermore, when the 4-methyl-1-pentene polymer is a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), the content of structural units derived from at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer is preferably 0 to 40 mol %, more preferably 2 to 20 mol %. The content of these structural units is calculated based on the total amount of repeating structural units in the 4-methyl-1-pentene polymer being 100 mol %. When the content of the structural units is within the above range, a uniform culture surface with excellent processability can be obtained, and the film has a good balance between toughness and strength, resulting in less deflection.

[0027] The 4-methyl-1-pentene polymer may contain structural units (hereinafter also referred to as "other structural units") other than the structural units derived from 4-methyl-1-pentene and the structural units derived from ethylene and the α-olefin having 3 to 20 carbon atoms, as long as the effects of the present invention are not impaired. The content of the other structural units is, for example, 0 to 10.0 mol%. When the 4-methyl-1-pentene polymer contains other structural units, the other structural units may be one type or two or more types.

[0028] Examples of monomers that derive other structural units include cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins. Examples of the cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins that can be used include the compounds described in paragraphs

[0035] to

[0041] of JP 2013-169685 A.

[0029] The 4-methyl-1-pentene polymers may be used alone or in combination of two or more.

[0030] Commercially available 4-methyl-1-pentene polymers can also be used. Specific examples include TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31, and DX845 (all trademarks) manufactured by Mitsui Chemicals, Inc. Also, 4-methyl-1-pentene polymers manufactured by other manufacturers that satisfy the above requirements can be preferably used. These commercially available products can be used alone or in combination of two or more.

[0031] 4-methyl-1-pentene polymers typically have a melting point of 200°C to 240°C and are highly heat-resistant. Furthermore, they do not undergo hydrolysis and have excellent water resistance, boiling water resistance, and steam resistance, allowing culture devices such as culture utensils containing 4-methyl-1-pentene polymers to be sterilized by high-pressure steam. 4-methyl-1-pentene polymers also have high visible light transmittance (typically 90% or higher) and do not emit autofluorescence, making culture utensils containing 4-methyl-1-pentene polymers easy to observe cultured cells. Furthermore, they exhibit excellent chemical resistance to most chemicals and are resistant to drug sorption, making them suitable for use in drug discovery screening and diagnostic applications. 4-methyl-1-pentene polymers are heat-sealable, allowing for easy thermal fusion not only between themselves but also with other materials. Furthermore, because they are thermoformable, they can be easily molded into culture utensils of any desired shape, including by imprinting or insert molding.

[0032] The weight-average molecular weight (Mw) of the 4-methyl-1-pentene polymer, measured by gel permeation chromatography (GPC) using standard polystyrene as the reference material, is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,000,000, and even more preferably 30,000 to 500,000. The sample concentration during GPC measurement can be, for example, 1.0 to 5.0 mg / ml. The molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer is preferably 1.0 to 30, more preferably 1.1 to 25, and even more preferably 1.1 to 20. Orthodichlorobenzene is preferably used as the solvent for GPC. Measurement conditions include, but are not limited to, those shown in the Examples below.

[0033] By setting the weight-average molecular weight (Mw) to the above upper limit or less, in the molding method of the 4-methyl-1-pentene polymer described below, the occurrence of defects such as gelling in the film produced by melt molding is easily suppressed, and a film with a uniform surface can be easily produced. Furthermore, when produced by a solution casting method, the solubility in a solvent is improved, and defects such as gelling in the film are easily suppressed, and a film with a uniform surface can be easily produced.

[0034] Furthermore, by setting the weight-average molecular weight (Mw) to the above lower limit or more, the culture material tends to have sufficient strength. Furthermore, by setting the molecular weight distribution within the above range, the stickiness of the surface of the culture material produced tends to be suppressed, and the toughness of the culture material tends to be sufficient, making it easier to suppress the occurrence of cracks during bending or cutting during molding.

[0035] When two or more types of 4-methyl-1-pentene polymers are used, the weight average molecular weight (Mw) and molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer may be within the above-mentioned ranges.

[0036] Because 4-methyl-1-pentene polymer has the excellent properties described above, a culture device in which at least the culture surface is formed from the culture component of the present invention does not adversely affect the culture, and also has good stability, light transmittance, moldability, and can be sterilized, making it an excellent material for culture components.

[0037] [Method of producing 4-methyl-1-pentene polymer] The method for producing the 4-methyl-1-pentene polymer may be any method capable of polymerizing 4-methyl-1-pentene, an olefin, or other monomers. Furthermore, a chain transfer agent, such as hydrogen, may be present in the presence of the polymer to control the molecular weight and molecular weight distribution. The equipment used for the production is not limited. The polymerization method may be a known method, such as a gas-phase method, a slurry method, a solution method, or a bulk method. The slurry method or the solution method is preferred. Furthermore, the polymerization method may be a single-stage polymerization method or a multi-stage polymerization method, such as a two-stage polymerization method, in which multiple polymers with different molecular weights are blended into the polymerization system. Whether single-stage or multi-stage polymerization is used, when hydrogen is used as a chain transfer agent, it may be added all at once or in portions, for example, at the initial, middle, or final stages of polymerization. The polymerization may be carried out at room temperature or, if necessary, heated. From the viewpoint of polymerization efficiency, the polymerization is preferably carried out at 20°C to 80°C, and particularly preferably at 40°C to 60°C. The catalyst used in the production is not limited, but from the viewpoint of polymerization efficiency, it is preferable to use, for example, a solid titanium catalyst component (I) described in WO 2006 / 054613 or an olefin polymerization catalyst (metallocene catalyst) containing a transition metal compound (A) described in WO 2014 / 050817.

[0038] When the culture medium is formed from a composition containing the 4-methyl-1-pentene polymer (X), the 4-methyl-1-pentene polymer (X) is preferably contained in an amount of 90% by mass or more but less than 100% by mass, more preferably 95% by mass or more but less than 100% by mass, and particularly preferably 99% by mass or more but less than 100% by mass, relative to 100% by mass of the culture medium. If the culture medium contains a large amount of components other than the 4-methyl-1-pentene polymer (X), not only will the oxygen permeability decrease, but the transparency and strength will also decrease.

[0039] The material forming the culture member of the present invention may contain components other than the 4-methyl-1-pentene polymer (X). Examples of components other than the 4-methyl-1-pentene polymer (X) include additives such as heat stabilizers, light stabilizers, processing aids, plasticizers, antioxidants, lubricants, antifoaming agents, antiblocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents.

[0040] [Water contact angle] The culture member of the present invention has a water contact angle on its culture surface that is greater than 100° and not greater than 160°. The water contact angle is preferably greater than 100° and not greater than 150°, more preferably greater than 100° and not greater than 130°, and even more preferably greater than 105° and not greater than 130°. If the water contact angle on the culture surface of the culture member is 100° or less, cells and the like will adhere to the culture member, making it difficult to form spheroids. Furthermore, if the water contact angle on the culture surface of the culture member is greater than 160°, contact between the culture surface and the medium will be insufficient, reducing the efficiency of oxygen supply to the medium.

[0041] The method for measuring the water contact angle is not particularly limited, and known methods can be used, but the sessile drop method is preferred. The water contact angle can be measured, for example, in accordance with Japanese Industrial Standard JIS-R3257 (test method for wettability of substrate glass surface), by dropping a water droplet of 4 μL or less, which can be considered as a sphere, under constant temperature and humidity conditions of 25±5° C. and 50±10% onto the surface of a culture member or a measurement sample made of the same material as the culture member, and measuring the angle of the contact interface between the measurement sample and the water droplet within 1 minute immediately after the water droplet contacts the surface of the measurement sample by the sessile drop method.

[0042] [Oxygen permeability] The oxygen permeability of the culture element of the present invention at a temperature of 23°C and humidity of 0% is 4500 to 90000 cm 3 / (m 2 × 24h × atm), preferably 4500 to 67500 cm 3 / (m 2 × 24h × atm), and more preferably 4500 to 47000 cm 3 / (m 2 × 24h × atm), and more preferably 4500 to 45000 cm 3 / (m 2 ×24h×atm).

[0043] If the oxygen permeability of the culture component is too low, the oxygen concentration in the culture medium will be low and cells will not grow sufficiently. On the other hand, if the oxygen permeability is too high, the oxygen concentration in the culture medium will be too high, and cell function will be reduced due to oxygen stress. When the oxygen permeability is within the above upper and lower limits, cells will maintain good morphology and can grow efficiently depending on the culture period.

[0044] The oxygen permeability coefficient [cm] at a temperature of 23°C and humidity of 0% was measured using a differential pressure gas permeability measurement method for a measurement sample made from the same material as the culture medium or the culture medium. 3 ×mm / (m 2 × 24h × atm)] was measured, and the oxygen permeability coefficient was divided by the thickness of the culture medium (μm) to obtain the oxygen permeability [cm 3 / (m 2× 24h × atm)]. The equipment used for the measurement is not particularly limited as long as it uses a differential pressure gas permeability measurement method, and an example is the differential pressure gas permeability measurement device MT-C3 manufactured by Toyo Seiki Seisakusho. The measurement sample is prepared by cutting a 90 × 90 mm test piece from a 50 μm thick film, for example, and the measurement section diameter is 70 mm (permeation area is 38.46 cm 2 Because the oxygen permeability is high, an aluminum mask is placed on the sample in advance to reduce the actual permeation area to 5.0 cm. 2 The culture member used for measuring oxygen permeability or the measurement sample made of the same material as the culture member may or may not have been subjected to microfabrication or surface modification treatment, but it is preferable that no treatment has been performed.

[0045] [cells, tissues, or organs] In this specification, cells, tissues, or organs are also simply referred to as "cells, etc." The origin of the cells, etc. is not particularly limited and may be any organism, such as an animal, a plant, an insect, a fungus, a protozoan, or a bacterium, but animals or plants are preferred, animals are more preferred, and mammals are particularly preferred. The culture device of the present invention allows cells, etc. to be cultured without adhesion and has excellent oxygen permeability, so it is preferable that the cells, etc. be non-adhesive.

[0046] The cells of the present invention are not particularly limited and include, for example, plant cells, animal cells, insect cells, etc., but are preferably animal cells, and more preferably mammalian cells. Mammalian cells are preferably cells derived from humans, monkeys, mice, rats, pigs, dogs, sheep, cats, or goats, and more preferably cells derived from humans. The cells may be cells cultured in two dimensions or three dimensions, and include spheroids obtained by cell culture. The cells of the present invention may be frozen or refrozen. Furthermore, the cells of the present invention may be passaged, and the number of passages is not particularly limited. Animal cells that can be used include normal cells, cancer cells, and fused cells such as hybridomas, and may also be cells that have been artificially treated, such as by gene transfer. Animal cells may be either primary cultured cells or established, passaged cells. Animal cells may be either suspension cells or adherent cells. Examples of animal cells include undifferentiated pluripotent stem cells, differentiated cells derived from pluripotent stem cells (including pluripotent stem cells that have initiated differentiation induction and are already in the process of differentiation), undifferentiated somatic stem cells, differentiated cells derived from somatic stem cells (including somatic stem cells that have initiated differentiation induction and are already in the process of differentiation), and differentiated cells derived from animal tissues.

[0047] In the present invention, differentiated cells are not limited to cells that have matured to the final stage of differentiation (terminally differentiated mature cells). The differentiated cells may be cells that differentiate and mature from ectoderm, mesoderm, or endoderm.

[0048] Pluripotent stem cells refer to cells that have the pluripotency to differentiate into all cells that make up a living organism and the ability to self-renew, maintaining this pluripotency even after cell division. Pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), muse cells (multi-lineage differentiating stress enduring cells), embryonic carcinoma cells (EC cells), trophoblast stem cells (TS cells), and epiblast stem cells (EpiS cells). Pluripotent stem cells are preferably ES cells or iPS cells, more preferably iPS cells.

[0049] ES cells can be established by isolating the inner cell mass from blastocysts of mammalian fertilized eggs and culturing the inner cell mass on fibroblast feeders. Subcultures can be maintained in a medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc. Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485, etc. Human ES cell lines, for example, WA01 (H1) and WA09 (H9) are available from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0050] iPS cells can be generated by introducing specific reprogramming factors into somatic cells in the form of DNA or protein (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); International Publication WO 2007 / 069666).

[0051] The somatic cells refer to all animal cells (preferably mammalian cells, including human cells) excluding germline cells such as eggs, oocytes, and ES cells, or totipotent cells, and include fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Examples of somatic cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; tissue progenitor cells; and differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts, hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells, brain cells, lung cells, kidney cells, and adipocytes.

[0052] The reprogramming factors may be composed of genes, their gene products, or non-coding RNAs that are specifically expressed in ES cells, genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products, or non-coding RNAs, or low-molecular-weight compounds. Examples of genes included in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination of two or more.

[0053] Somatic stem cells refer to cells that have a limited differentiation potential, which allows them to differentiate into specific cells, and the ability to self-renew, which allows them to maintain that limited differentiation potential even after cell division. Somatic stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, etc.

[0054] Examples of differentiated cells derived from animal tissues include various precursor cells, such as adipocytes, liver cells, kidney cells, pancreatic cells, mammary gland cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, cardiac muscle cells, nerve cells, glial cells, dendritic cells, chondrocytes, osteoblasts, osteoclasts, osteocytes, fibroblasts, various blood cells, retinal cells, corneal-derived cells, gonad-derived cells, and various gland cells.

[0055] The cells may be used singly or in combination of two or more types.

[0056] Because animal cells are suitable for spheroid formation, preferably pluripotent stem cells and differentiated cells derived from pluripotent stem cells, more preferably iPS cells and differentiated cells derived from iPS cells, even more preferably iPS cell-derived cardiomyocytes, and particularly preferably iPS cell-derived human cardiomyocytes. Alternatively, because animal cells are suitable for spheroid formation, preferably somatic stem cells, more preferably mesenchymal stem cells. Alternatively, because animal cells are suitable for spheroid formation, preferably cancer cells. Alternatively, because the state of the cells can be more closely resembled to the state in vivo with a three-dimensional structure, preferably animal cells that are generally required to be cultured in three dimensions, more preferably cells that form spheroids, such as hepatocytes, neurons, cardiomyocytes, pancreatic beta cells, vascular endothelial cells, adipocytes, adipose-derived stem cells, chondrocytes, mesenchymal stem cells, hair follicle epithelial stem cells, hair papilla cells, skin fibroblasts, skin keratinocytes, and osteoblasts.

[0057] When the animal cells are differentiated cells derived from pluripotent stem cells, the cells can be prepared from pluripotent stem cells by a known differentiation induction method. Alternatively, commercially available differentiated cells derived from pluripotent stem cells may be used. When the animal cells are somatic stem cells, cells collected from animals or commercially available cells may be used.

[0058] The differentiated cells derived from pluripotent stem cells are preferably cardiomyocytes derived from iPS cells.

[0059] iPS cell-derived cardiomyocytes can be produced, for example, by the well-known protein-free cardiac differentiation (PFCD) method (see International Publication No. 2015 / 182765). Because the protein-free cardiac differentiation (PFCD) method can achieve high cardiac differentiation efficiency, the iPS cell-derived cardiomyocytes produced by this method can achieve high cardiomyocyte purity.

[0060] In the present invention, a spheroid refers to a mass of cells, and can be referred to as a cell aggregate or cell mass. The spheroid may be a spheroid containing a single cell, such as a cardiomyocyte, or a spheroid containing two or more different cell types, such as various fibroblasts or vascular endothelial cells and cardiomyocytes. Usable cells include the various cells described above. Spheroids formed using the culture member or culture device of the present invention are preferably spheroids containing pluripotent stem cells and spheroids containing differentiated cells derived from pluripotent stem cells, more preferably spheroids containing iPS cells or spheroids containing differentiated cells derived from iPS cells, and even more preferably spheroids containing cardiomyocytes derived from iPS cells.

[0061] The spheroids preferably have a differentiated cell purity (number of differentiated cells / number of cells constituting the spheroid × 100) of 10% or more, more preferably 13% or more, even more preferably 15% or more, even more preferably 20% or more, and particularly preferably 30% or more. When iPS cells are induced to differentiate into cardiomyocytes as target cells to form spheroids, the spheroids contain iPS cell-derived cardiomyocytes at a cardiomyocyte purity of preferably 10% or more, more preferably 20% or more, even more preferably 30% or more. The number of differentiated cells can be determined by known methods, for example, by flow cytometry analysis using antibodies against cardiac markers such as cardiac troponin T (cTnT), troponin, myosin regulatory light chain 2 (MYL2), and myosin regulatory light chain 7 (MYL7). The number of cells constituting the spheroids can be determined by known methods, for example, by treating the spheroids with trypsin to dissociate them into single cells and then counting the number of single cells.

[0062] When pluripotent stem cells are differentiated to obtain differentiated cells, the resulting spheroids will contain not only the differentiated cells of interest, but also a considerable number of undifferentiated cells and cells that have differentiated in a manner other than the desired one. Therefore, if the differentiated cell purity is within the above range, the reliability of the data obtained when the differentiated cell spheroids are used in various tests will be high, and the reproducibility will also be high.

[0063] The size of the spheroids is not particularly limited. Although it varies depending on the cell type, cell number, medium, and culture period, the size of the spheroids is preferably, for example, an average diameter of 10 to 10,000 μm, more preferably 10 to 8,000 μm, and even more preferably 10 to 5,000 μm. The diameter of the spheroids can be determined, for example, by observing them under a microscope and measuring the diameter on a photograph, or by using a particle size distribution analyzer.

[0064] The number of cells constituting the spheroid is not particularly limited. It varies depending on the cell type forming the spheroid, the medium, the number of days of culture, etc., but for example, 1 × 10 cells per spheroid 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, 1×10 8 pcs or more, 1×10 9 The number of cells constituting the spheroid can be calculated, for example, from the fluorescence intensity after staining the cells with a fluorescent reagent and a calibration curve of the cell number and fluorescence intensity.

[0065] The medium used for cell culture can be selected appropriately depending on the cells. The type of medium is not particularly limited, but any basal cell culture medium, differentiation medium, or medium specifically designed for primary culture can be used. Specific examples include Essential 8, Eagle's Minimal Essential Medium (EMMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI 1640, Ham's F-12, MCDB medium, Williams' Medium E, Hepatocyte thaw medium, and mixtures thereof. However, these are not limited to these. Any medium containing the components necessary for cell proliferation and differentiation can be used. Furthermore, media supplemented with serum, various growth factors, differentiation inducers, antibiotics, hormones, amino acids, sugars, salts, etc. may also be used. The culture temperature is also not particularly limited, but is typically around 25–40°C. The amount of the medium is not particularly limited, but the height of the medium is preferably 3 to 30 mm, more preferably 3 to 25 mm, and even more preferably 4 to 20 mm.

[0066] In the present invention, the term "tissue" refers to a collection of similar cells that perform a similar function, and is a different concept from spheroids. The tissue is not particularly limited, and examples include epithelial tissue, connective tissue, muscle tissue, and nervous tissue. The tissue is preferably a tissue containing cells that form spheroids, and examples of such tissues include nervous tissue containing nerve cells, cardiac tissue containing cardiomyocytes, adipose tissue containing adipocytes or adipose-derived stem cells, cartilage tissue containing chondrocytes, and bone tissue containing osteoblasts. Among these, the tissue is preferably nervous tissue or cardiac tissue, with cardiac tissue being more preferred, due to its high oxygen requirement. Furthermore, the tissue is preferably a tissue containing somatic stem cells, as it is suitable for spheroid formation.

[0067] In the present invention, an organ refers to a group of tissues that work together to achieve a common goal. The organ is not particularly limited, and examples include the lungs, heart, liver, kidneys, spleen, pancreas, gallbladder, esophagus, stomach, skin, and brain. The organ is preferably an organ containing cells that form spheroids, and examples of such organs include the liver containing hepatocytes, the pancreas containing pancreatic β cells, blood vessels containing vascular endothelial cells, bone marrow containing mesenchymal stem cells, hair follicles containing hair follicle epithelial stem cells or dermal papilla cells, kidneys, and skin containing skin fibroblasts or skin keratinocytes. Among these, the skin, kidneys, liver, pancreas, heart, and hair follicles are preferred due to their high oxygen requirements, with the heart being even more preferred. Furthermore, the organ is preferably an organ containing somatic stem cells, as they are suitable for spheroid formation.

[0068] [Culture equipment] In the present invention, a culture instrument refers to any instrument used for culturing cells, etc. At least a portion of the culture instrument is composed of the culture member. The culture instrument may be composed entirely of the culture member, or only a portion of the culture instrument may be composed of the culture member. When only a portion of the culture instrument is composed of the culture member, at least the culture surface on which cells, etc. are cultured is composed of the culture member of the present invention.

[0069] The culture device is typically used in an apparatus such as an incubator, a mass culture apparatus, or a perfusion culture apparatus.

[0070] The culture tool may be any of various known culture tools, and is not particularly limited in shape or size. Examples of the culture tool include culture vessels such as dishes, flasks, plates, bottles, bags, and tubes, as well as inserts, cups, insoles, and slides, with culture vessels being preferred.

[0071] The culture device may be a culture device having at least one well or a culture vessel having at least one well. A culture vessel having at least one well is, for example, a plate having at least one well, more specifically, a plate having 6, 12, 24, 48, 96, 384, 1536, or other wells. Generally, culture devices having a well-like depression on the bottom surface require a thick bottom to stabilize the complex shape of the bottom, making it difficult to adequately supply oxygen to cells, etc. By using the culture member of the present invention, even plates having 1, 6, 12, 24, 48, 96, 384, 1536, or other wells maintain a stable shape and provide sufficient oxygen to cells, etc.

[0072] The culture tool is preferably a tool whose bottom surface serves as a culture surface in order to hold or store the culture medium. When the culture tool is a dish, flask, insert, or plate, the bottom surface serves as the culture surface, and the culture component of the present invention preferably constitutes at least a part or all of the bottom, side, and top surfaces of these. When at least the bottom surface (culture surface) is constituted by the culture component of the present invention, oxygen can be more efficiently supplied to the culture medium through the culture component, allowing cells and the like in the culture medium to proliferate more efficiently. Furthermore, cells can be cultured at a higher density while maintaining their function.

[0073] The shape of the bottom of the culture device is not particularly limited, and examples include a flat bottom, a round bottom (U-bottom), a flat bottom (F-bottom), a conical bottom (V-bottom), and a flat bottom with a curved edge. When processing into a round bottom (U-bottom), a flat bottom (F-bottom), a conical bottom (V-bottom), a flat bottom with a curved edge, etc., the bottom may be processed in one step by general injection molding or press molding, or it may be produced by first preparing a film or sheet and then performing secondary processing such as vacuum forming or pressure forming. The shape of the bottom is selected depending on the purpose of the culture, but a flat bottom is usually desirable for two-dimensional culture of cells, etc., and a round bottom (U-bottom) or conical bottom (V-bottom) is usually desirable for three-dimensional culture.

[0074] The portions of the culture instrument other than the culture member may be made of a material other than the culture member. The material other than the culture member is not particularly limited, and known materials can be used. Examples of such materials include polystyrene, polydimethylsiloxane (PDMS), cyclic olefin polymers, cyclic olefin copolymers, and glass.

[0075] The culture device may be used after its culture surface and / or portions other than the culture surface are coated with a natural polymer material, a synthetic polymer material, or an inorganic material. Coating the culture surface of a culture device with a natural polymer material, a synthetic polymer material, or an inorganic material improves the proliferation of cells, etc., but also makes it easier for cells, etc., to adhere to the culture surface. Whether or not the culture surface is coated can be determined depending on the type of cells, etc., but it is usually preferable not to coat it.

[0076] The culture instrument of the present invention may be disinfected or sterilized to prevent contamination. The disinfection or sterilization method is not particularly limited, and examples include physical disinfection methods such as steam circulation, boiling, intermittent irradiation, and ultraviolet light; chemical disinfection using gases such as ozone or disinfectants such as ethanol; heat sterilization methods such as high-pressure steam and dry heat; irradiation sterilization methods such as gamma ray sterilization, electron beam sterilization, and high-frequency sterilization; and gas sterilization methods such as ethylene oxide gas sterilization and hydrogen peroxide gas plasma sterilization. Among these, ethanol disinfection, high-pressure steam sterilization, gamma ray sterilization, electron beam sterilization, and ethylene oxide gas sterilization are preferred because of their simple operation and ability to achieve sufficient sterilization. These disinfection or sterilization methods may be performed alone or in combination of two or more.

[0077] The method for manufacturing the culture instrument of the present invention is not particularly limited, and when the entire culture instrument is composed of the culture member, it can be manufactured by the same method as the method for manufacturing the culture member. When only a part of the culture instrument is formed from the culture member, the culture instrument can be obtained by appropriately joining the culture member and other members. The joining method is not particularly limited, and the culture member and other members may be formed integrally, or may be adhered to each other via an adhesive or pressure-sensitive adhesive.

[0078] The culture instrument of the present invention is preferably a culture instrument for spheroid formation, and more preferably a cell culture instrument for spheroid formation.

[0079] [Culture method] The method for culturing cells and the like of the present invention is a culture device for culturing cells, tissues, or organs on its culture surface, the culture device containing 4-methyl-1-pentene polymer (X), the culture surface having a water contact angle of more than 100° and not more than 160°, and an oxygen permeability of 4500 to 90000 cm at a temperature of 23°C and a humidity of 0%. 3 / (m 2 The method for culturing cells, tissues, or organs includes the steps of: (A) bringing cells, tissues, or organs into contact with a culture surface of a culture device formed from the culture device, or at least the culture surface of which is a culture element having a size of 1000×24h×atm; and (B) culturing the cells, tissues, or organs in contact with the culture surface to form spheroids.

[0080] In the culture method of the present invention, when cells or the like are cultured on the culture surface of the culture member or the culture instrument, the cells or the like can efficiently form spheroids. Furthermore, according to the present invention, pluripotent stem cells can be easily differentiated efficiently into differentiated cells.

[0081] [Process (A)] The method for contacting cells or the like with the culture surface of a culture member or culture instrument is not particularly limited as long as it allows the cells or the like to come into contact with the culture surface of the culture member or culture instrument, and examples thereof include seeding cells or the like on the culture surface of the culture member or culture instrument. More specifically, for example, cells or the like suspended in a medium are added to a culture vessel using a pipette or the like, and the culture vessel is shaken as necessary to distribute the cells evenly throughout the culture vessel, and then the vessel is left to stand in an incubator.

[0082] The seeding density of cells is not particularly limited as long as the cells can grow and differentiate. When the cells are undifferentiated pluripotent stem cells or differentiated cells derived from pluripotent stem cells, the seeding density is preferably 0.1 × 10 5 cells / cm 2 ~10.0×10 5 cells / cm 2 and more preferably 0.3 × 10 5 cells / cm 2 ~5.0×10 5 cells / cm 2 and more preferably 0.5 × 10 5 cells / cm 2 ~3.0×10 5 cells / cm 2 is. A cell seeding density within the above range is preferable because cell proliferation and differentiation can be carried out more efficiently than when the density is outside the above range.

[0083] The medium used for seeding is not particularly limited as long as it is a medium in which cells etc. can survive, and may be appropriately selected depending on the cells etc. to be used. The medium used for seeding may be the medium used in step (B) described below. When the cells are undifferentiated pluripotent stem cells or differentiated cells derived from pluripotent stem cells, the medium used for seeding can be, for example, any basal cell culture medium, differentiation medium, or medium specifically for primary culture, such as Essential 8, StemFit medium, ReproFF2 medium, Stem-PartnerSF, Eagle's Minimal Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham's F-12, MCDB medium, Williams' Medium E, and mixtures thereof. Furthermore, media supplemented with serum, various growth factors, differentiation inducers, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, etc. may also be used.

[0084] The amount of medium used for seeding is not particularly limited, but the height of the medium added to the culture vessel is preferably 3 to 30 mm, more preferably 3 to 25 mm, and even more preferably 4 to 20 mm. The culture temperature is not particularly limited, but is usually about 25 to 40°C.

[0085] [Process (B)] The method for culturing cells etc. in contact with the culture surface to form spheroids is not particularly limited as long as it is possible to supply oxygen, nutrients etc. to the cells etc. in contact with the culture surface and culture the cells etc. to form spheroids. For example, oxygen may be supplied to a culture incubator containing a culture device containing medium, and oxygen may be supplied to the cells etc. via the culture member, and the temperature may be maintained at 37°C for a certain period of time.

[0086] The medium used for cell culture can be selected appropriately depending on the cells. The type of medium is not particularly limited, but any basal cell culture medium, differentiation medium, or medium specifically designed for primary culture can be used. Specific examples include Essential 8, Eagle's Minimal Essential Medium (EMMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI 1640, Ham's F-12, MCDB medium, Williams' Medium E, Hepatocyte thaw medium, and mixtures thereof. However, these are not limited to these. Any medium containing the components necessary for cell proliferation and differentiation can be used. Furthermore, media supplemented with serum, various growth factors, differentiation inducers, antibiotics, hormones, amino acids, sugars, salts, etc. may also be used. The culture temperature is also not particularly limited, but is typically around 25–40°C. The amount of the medium is not particularly limited, but the height of the medium is preferably 3 to 30 mm, more preferably 3 to 25 mm, and even more preferably 4 to 20 mm.

[0087] The culture period may be appropriately selected depending on the type, size, degree of differentiation, etc. of the cells and the spheroids to be formed. When forming spheroids containing iPS cell-derived cardiomyocytes, the culture period is preferably 7 to 30 days, more preferably 10 to 25 days, and preferably 11 to 20 days.

[0088] [Process (C)] The method for culturing cells or the like of the present invention may further comprise the step (C) of inducing differentiation of the cells or the like. The method for inducing differentiation of the cells etc. is not particularly limited, and differentiation into the desired differentiated cells may be induced according to known protocols, and commercially available differentiation-inducing media or differentiation kits may be used.

[0089] The conditions for inducing differentiation are not particularly limited and can be appropriately set depending on the cell type used, the target differentiated cell type, etc. Typically, differentiation can be induced by culturing in a culture medium containing a predetermined concentration of a predetermined cytokine, growth factor, or other compound.

[0090] When the method for culturing cells etc. of the present invention includes step (C), the cells etc. are preferably undifferentiated pluripotent stem cells, differentiated cells derived from pluripotent stem cells (including pluripotent stem cells that have started to be induced to differentiate and are already in the process of differentiation), undifferentiated somatic stem cells, or differentiated cells derived from somatic stem cells (including somatic stem cells that have started to be induced to differentiate and are already in the process of differentiation), more preferably undifferentiated pluripotent stem cells or differentiated cells derived from pluripotent stem cells, and even more preferably undifferentiated iPS cells or differentiated cells derived from iPS cells.

[0091] The differentiated cells of interest are not particularly limited and may be any differentiated cells, including various precursor cells, such as adipocytes, hepatocytes, kidney cells, pancreatic cells, mammary gland cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, cardiomyocytes, nerve cells, glial cells, dendritic cells, chondrocytes, osteoblasts, osteoclasts, osteocytes, fibroblasts, various blood cells, retinal cells, corneal-derived cells, gonad-derived cells, and various gland cells.

[0092] The differentiated cells of interest are preferably cells that form spheroids, more preferably cells with a high oxygen requirement. The differentiated cells of interest are preferably hepatocytes, nerve cells, cardiomyocytes, pancreatic β cells, vascular endothelial cells, adipocytes, adipose-derived stem cells, chondrocytes, mesenchymal stem cells, hair follicle epithelial stem cells, dermal papilla cells, skin fibroblasts, skin keratinocytes, osteoblasts, hematopoietic progenitor cells, etc., more preferably cardiomyocytes, nerve cells, or hepatocytes, and even more preferably cardiomyocytes.

[0093] Step (C) is preferably a step of inducing differentiation of pluripotent stem cells into cardiomyocytes, and more preferably a step of inducing differentiation of iPS cells into cardiomyocytes.

[0094] The method for inducing differentiation of pluripotent stem cells into cardiomyocytes is not particularly limited. Various methods are known for inducing differentiation of pluripotent stem cells into cardiomyocytes (e.g., Burridge et al., Cell Stem Cell. 2012 Jan 6;10(1):16-28; Kattman et al., Cell Stem Cell 2011;8:228-240; Zhang et al., Circ Res 2012;111:1125-1136; Lian et al., Nat Protoc 2013;8:162-175; WO 2016 / 076368; WO 2013 / 111875; Minami et al., Cell Rep. 2012,2(5):1448-1460, etc.), and examples of such methods include embryoid body formation, monolayer differentiation culture, and forced aggregation. In either method, the induction efficiency can be increased by sequentially applying mesoderm induction factors (e.g., activin A, BMP4, bFGF, VEGF, SCF, etc.), cardiac determination factors (e.g., VEGF, DKK1, Wnt signal inhibitors (e.g., IWR-1, IWP-2, IWP-4, etc.), BMP signal inhibitors (e.g., NOGGIN, etc.), TGFβ / activin / NODAL signal inhibitors (e.g., SB431542, etc.), retinoic acid signal inhibitors, etc.), cardiac differentiation factors (e.g., VEGF, bFGF, DKK1, etc.), etc.

[0095] The method for inducing differentiation of iPS cells into cardiomyocytes is not particularly limited, and for example, the well-known protein-free cardiac differentiation (PFCD) method can be used (see International Publication No. 2015 / 182765). Because the protein-free cardiac differentiation (PFCD) method can achieve high cardiac differentiation efficiency, step (C) is preferably a step of inducing differentiation of iPS cells into cardiomyocytes by the protein-free cardiac differentiation (PFCD) method.

[0096] Step (C) may be performed after step (A) and before step (B), or may be performed simultaneously with step (B), or may be performed only during a portion of the period during which step (B) is performed. Step (C) is preferably performed simultaneously with step (B), since this allows for more efficient induction of differentiation of pluripotent stem cells.

[0097] The differentiation induction period in step (C) can be appropriately set depending on the type of cells used, the type of cells to be differentiated, the degree of differentiation, the differentiation induction method, etc. When inducing differentiation of iPS cells into cardiomyocytes, the differentiation induction period is preferably 7 to 30 days, more preferably 10 to 25 days, and even more preferably 11 to 20 days.

[0098] [Characteristics of the culture method] The method for culturing cells etc. of the present invention is preferably a method for culturing iPS cells or differentiated cells derived from iPS cells, and more preferably a method for culturing cardiomyocytes derived from iPS cells.

[0099] The method for culturing cells etc. of the present invention preferably increases cardiac muscle purity. The increase refers to a myocardial purity that is higher than that of a control when pluripotent stem cells are differentiated into cardiomyocytes under experimental conditions that are the same as those of the method for culturing cells of the present invention, except that a polystyrene culture member or culture utensil is used instead of the culture member or culture utensil. The myocardial purity is preferably 1.3 times or more higher than that of the control, more preferably 2 times or more higher, and even more preferably 3 times or more higher.

[0100] The myocardial purity is the percentage (%) of the number of cardiomyocytes differentiated from pluripotent stem cells to the total number of differentiated cells derived from the pluripotent stem cells. The method for measuring the myocardial purity is not particularly limited, but for example, after a predetermined period of differentiation induction, the total number of cells and the number of cells expressing cardiomyocyte markers can be measured and the percentage can be calculated. When the cells form spheroids, the spheroids can be separated into single cells by a known method, and the total number of cells constituting the spheroids can be used as the total cell number. For measuring the number of cells, for example, a hemocytometer, FACS, or the like can be used.

[0101] The cardiomyocyte marker is not particularly limited, and examples thereof include cardiac troponin C (cTnC), cardiac troponin I (cTnI), cardiac troponin T (cTnT), myosin light chain, αActinin, NKX2.5, KCNQ1, HERG1b, Cav1.2, Nav1.5, etc. The cardiomyocyte marker is preferably cardiac troponin T (cTnT). The number of cells expressing a cardiomyocyte marker can be measured, for example, by FACS using an antibody against the cardiomyocyte marker. The myocardial purity can be preferably calculated by the formula: number of cTnT-positive cells / total number of cells constituting the spheroid×100.

[0102] The method for culturing cells etc. of the present invention preferably increases the expression of the MYL2 (myosin regulatory light chain 2) gene in cardiomyocytes. The increase refers to a state in which pluripotent stem cells are differentiated into cardiomyocytes using a polystyrene culture member or culture utensil instead of the culture member or culture utensil under experimental conditions identical to those of the method for culturing cells of the present invention, and the expression level of the MYL2 gene in the cardiomyocytes is higher than that of a control, preferably two-fold or more, and more preferably three-fold or more, of the control. The expression level of the MYL2 gene can be measured by known methods, such as quantitative RT-PCR.

[0103] The method for culturing cells etc. of the present invention preferably increases the pulsation rate of cardiomyocytes. The increase refers to a higher pulsation rate of cardiomyocytes than a control when pluripotent stem cells are differentiated into cardiomyocytes under experimental conditions identical to those of the method for culturing cells of the present invention, except that a polystyrene culture member or culture utensil is used instead of the culture member or culture utensil. The pulsation rate of cardiomyocytes is preferably at least two times higher than that of the control, and more preferably at least three times higher. The pulsation rate of cardiomyocytes can be measured by known methods; for example, a video of cardiomyocytes can be taken and the BPM (beats per minute) of any cardiomyocyte can be measured.

[0104] [Spheroid] The spheroids of the present invention are formed by the above-described culture method. The spheroids of the present invention are characterized by a tendency for high uniformity in size and shape due to oxygen supply to the interior of the cells during culture. Furthermore, the cells contained in the spheroids of the present invention retain normal functions and have high cell purity, which increases the reliability of data when the spheroids are used in various tests and also increases reproducibility. The spheroids of the present invention can be suitably used for evaluating cell function, screening drugs, and as a cell source for regenerative medicine and cell therapy. [Example]

[0105] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0106] [Measurement of weight average molecular weight (Mw) and molecular weight distribution (Mw / Mn)] The weight average molecular weight Mw and molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer used in the examples were measured by gel permeation chromatography (GPC). Specifically, the weight average molecular weight (Mw) and number average molecular weight (Mn) of the polymer dissolved in orthodichlorobenzene were measured under the following conditions, with the molecular weight calibrated using standard polystyrene. Apparatus: Gel permeation chromatograph HLC-8321 GPC / HT type (Tosoh Corporation) Data analysis software: Empower3 (Waters) Detector: Differential refractometer - Serially connected columns: TSKgel GMH6-HT (2 columns) and TSKgel GMH6-HTL (2 columns) Column temperature: 140℃ ·Flow rate: 1.0ml / min Sample concentration: 1.5mg / ml

[0107] [Measurement of hanging distance] A test piece measuring 100 mm x 10 mm was cut from the film of the Production Example or the culture vessel of the Comparative Example, and fixed to the test stand with 50 mm of the test piece extending horizontally from the horizontal upper surface of the test stand. Three minutes after fixation, the distance that the tip of the test piece extending from the test stand hung vertically downward from the horizontal plane including the upper surface of the test stand was measured. The temperature from fixation to measurement was 23°C. The results are shown in Table 1.

[0108] [Presence or absence of deflection] On day 19 after differentiation induction, the culture vessel was removed from the incubator, and the bottom of the vessel was examined from the side to see if the film had sagged in the culture environment. If there was no change from when the vessel was made and no sagging of the film was observed, it was evaluated as "no sagging," and if there was a change from when the culture vessel was made and sagging of the film was observed, it was evaluated as "sagging."

[0109] [Water contact angle measurement] The water contact angle was measured in accordance with Japanese Industrial Standard JIS-R3257 (Testing Method for Wettability of Substrate Glass Surfaces). Under constant temperature and humidity conditions of 25±5°C and 50±10%, a water droplet of 4 μL or less, which can be considered spherical, was dropped onto the surface of the measurement sample, and the angle of the contact interface between the measurement sample and the water droplet was measured using the sessile drop method within one minute after the water droplet contacted the surface of the measurement sample.

[0110] [Oxygen permeability measurement] The oxygen permeability coefficient of the measurement sample was measured using a differential pressure gas permeability measuring device MT-C3 manufactured by Toyo Seiki Seisakusho Co., Ltd. under an environment of 23°C temperature and 0% humidity. The measurement section diameter was 70 mm (permeation area was 38.46 cm). 2 Because the oxygen permeability coefficient was expected to be large, an aluminum mask was placed on the sample in advance to reduce the actual permeation area to 5.0 cm. 2 It was decided. The measured oxygen permeability coefficient [cm 3 ×mm / (m 2 × 24h × atm)] by the thickness (μm) of the film (culture material) to obtain the oxygen permeability [cm 3 / (m 2 × 24h × atm)] was calculated.

[0111] [Manufacturing Example 1] Manufacturing of culture material TPX (registered trademark), a 4-methyl-1-pentene polymer (manufactured by Mitsui Chemicals, Inc.: molecular weight (Mw) = 428,000, molecular weight distribution (Mw / Mn) = 4.1), was used and fed into a T-die extruder equipped with a full-flight screw for extruding the substrate layer. The extrusion temperature was set to 270°C, the roll temperature to 60°C, and the roll rotation speed was varied to obtain a 50 μm thick film. The water contact angle and oxygen permeability of the obtained film were measured. The results are shown in Table 1.

[0112] [Production Example 2] Preparation of culture vessel The film was cut into 8 cm x 12 cm pieces and attached to the bottom of a 24-well polystyrene (PS) container frame using a medical adhesive (manufactured by 3M) to prepare a 24-well culture plate. The plate was then packed in a gamma-ray-resistant bag and sterilized by irradiating with 10 kGy of gamma rays.

[0113] [Example 1] Induction of differentiation from iPS cells to cardiomyocytes Using the culture vessel prepared in Production Example 2, differentiation of iPS cells into cardiomyocytes was induced by the method described below to form spheroids. The medium was used so that the medium height became 5 mm (medium volume: 1 mL).

[0114] [Example 2] Cultivation was carried out in the same manner as in Example 1, except that the amount of medium was increased to 10 mm (medium volume: 1.8 mL).

[0115] [Comparative Example 1] A culture vessel made of ultra-low adhesion PS with a culture surface thickness of 1000 μm (Corning, Costar TM Culture was carried out in the same manner as in Example 1, except that a 24-well plate (3473, with an inner diameter of the culture surface per well of 15 mm, 24-well plate) was used. The ultra-low adhesion surface PS culture vessel was a commercially available PS culture vessel (water contact angle 61°) that had been hydrophilized. The results of measuring the water contact angle are shown in Table 1.

[0116] Comparative Example 2 Cultivation was carried out in the same manner as in Comparative Example 1, except that the amount of medium was increased to 10 mm.

[0117] [iPS cell culture] <Preparation of medium and reagents> Human iPS cells (provided by the Institute for iPS Cell Research and Application) that had been frozen and stored in liquid nitrogen were used. The reagents used are as follows: Essential 8 (product number A1517001, Gibco) iMatrix-511 silk (product number 387-10131, Nippi) CultureSure TM Y-27632 (product number 034-24024, Fujifilm Wako Pure Chemical Industries, Ltd.) 0.5 mol / L-EDTA solution (pH 8.0) (product number 06894-85, Nacalai Tesque) PBS(-) (Product No. 166-23555, Fujifilm Wako Pure Chemical Industries, Ltd.) 2.5% Trypsin (product number 15090-046, Gibco) Trypan blue (product number 145-0022, Bio-Rad) Formaldehyde solution (product number 064-00406, Fujifilm Wako Pure Chemical Industries, Ltd.) Anti-Troponin TC (CT3) (Product No. SC-20025, Santacruz) Anti-Mouse IgG(H+L) Alexa Fluor 647 (Product No. A-21236, Thermo Fisher Scientific) Saponin from Soybeans (product number 192-08851, Fujifilm Wako Pure Chemical Industries, Ltd.) Cardiac differentiation induction medium (low molecular weight compound group A, low molecular weight compound group B): Follow the description in paragraphs

[0064] and

[0065] of WO 2015 / 182765.

[0118] The medium and reagents were prepared as follows. iPS cell medium: Essential 8 was brought to room temperature before use. CultureSure medium was used for thawing and subculture. TM Y-27632 was added to a concentration of 3 μM. Cardiac differentiation induction medium: When used, the cardiac differentiation induction medium was mixed with low molecular weight compound group A or low molecular weight compound group B and heated to 37°C before use. Stripping solution: 0.5 mol / L-EDTA solution (pH 8.0) adjusted to 0.5 mM with PBS(-). Warm to 37°C before use. · Y-27632 solution: Adjusted to 10 mM with PBS(-). · Singulation solution: 2.5% Trypsin adjusted to 0.25% with PBS(-). When used, it was heated to 37 °C before use. · Saponin solution: Saponin from Soybeans adjusted to a 0.1% solution with PBS(-). · Storage conditions: The Y-27632 solution, singulation solution, and other reagents necessary for cardiomyocyte culture were stored frozen, and the medium and dissociation solution were stored refrigerated.

[0119] <Culture schedule> Cell culture was performed according to the following schedule. iPS cells at passage 5 after thawing were used for differentiation induction. Sampling was performed on day 12 when cardiomyocyte beating was globally confirmed and on day 19 when they were more matured. Day-1: Reagent preparation and preparation Day0: Thawing of iPS cells Day3~: Passage of iPS cells (a total of 5 passages on days 3, 6, 10, 15, 19) Day23: Initiation of cardiomyocyte differentiation induction of iPS cells (day 0) Day45: Sampling and single cell isolation (FACS, gene expression analysis) on day 12 of cardiomyocyte differentiation induction Day39: Sampling and single cell isolation (FACS, gene expression analysis) on day 19 of cardiomyocyte differentiation induction

[0120] <iPS cell culture method> Thawing of iPS cells 1) Before thawing iPS cells, 10 mL of medium was placed in a tube and heated in a 37 °C water bath. 2) The frozen iPS cell tube was made semi-molten in a 37 °C water bath and slowly transferred to a tube containing 10 mL of pre-warmed medium in a clean bench. 3) After centrifugation at 100×g for 3 minutes at room temperature, the supernatant was removed. 4) 10 mL of fresh medium was added and gently mixed, 0.13 μg / cm 2iMatrix-511 silk was added to the cell suspension, and the cells were seeded onto a 10 cm dish and cultured at 37°C in a 5% CO2 incubator.

[0121] Passaging of iPS cells 1) The state of the cells was observed under a phase contrast microscope, and the split ratio for passage was determined to be between 1:12 and 1:20. 2) The culture supernatant was collected in a tube. After washing with PBS(-), a detachment solution was added and the mixture was incubated at 37°C in a 5% CO2 incubator for approximately 5 minutes. 3) After the reaction, the dish was tilted to detach the cells, and the cells were collected into a tube using the collected supernatant. 4) After centrifugation at 100×g at room temperature for 3 minutes, the supernatant was removed. 5) Add 10 mL of fresh medium, mix gently, and add 0.13 μg / cm 2 iMatrix-511 silk was added to the cell suspension, and the cells were seeded onto a 10 cm dish and cultured at 37°C in a 5% CO2 incubator.

[0122] <Method for inducing cardiac differentiation> An outline of the experimental schedule is shown in Figure 1. Differentiation of iPS cells into cardiomyocytes was induced using the protein-free cardiac differentiation (PFCD) method. Day 0: Approximately 50% confluent iPS cells were detached using a detachment solution and centrifuged at 100 × g at room temperature for 3 minutes. After removing the supernatant, the cells were suspended in fresh medium and transferred to untreated 55cm 2 The cells were seeded onto a dish and left to stand for 4 hours in a 37°C, 5% CO2 incubator. Then, the cells were collected into a tube and replaced with a cardiac differentiation-inducing medium containing low molecular weight compound group A. 2 × 10 5 Cells were seeded at 1000 cells / well and cultured at 37°C in a 5% CO2 incubator. Experiments were performed in triplicate. Day 3: The medium was replaced with a cardiac differentiation-inducing medium containing low molecular weight compound group B. Day 5: The medium was replaced with a cardiac differentiation-inducing medium containing low molecular weight compound group B. After the 7th day: The medium was changed every 3 to 4 days with a cardiomyocyte differentiation induction medium. Additionally, the wells were changed during the medium exchanges on the 3rd and 7th days of differentiation induction.

[0123] <Single-cell formation of cell aggregates (sampling on the 12th and 19th days of differentiation induction)> 1) The cell aggregates (presumed to be myocardial masses) were collected into a tube, and the supernatant was removed after the cell aggregates had sedimented naturally. 2) 1 mL of a single-cell formation solution was added, and it was heated in a 37°C water bath for about 30 minutes and stirred until the cell aggregates became single cells. 3) The reaction was stopped by diluting with 3 mL of PBS(-) at three times the volume. 4) The cell count of the cell suspension was performed and used for FACS or RNA extraction described below.

[0124] <Preparation of FACS samples> 1×10 6 single cells after singulation were transferred to a tube, centrifuged at 300×g for 5 minutes at room temperature, and then the supernatant was removed and resuspended with 1 mL of PBS(-). Formaldehyde (FUJIFILM Wako Pure Chemical Corporation) was added to the cell suspension to a final concentration of 4%, and it was allowed to stand at room temperature for 5 minutes. After centrifuging at 300×g for 3 minutes at room temperature and removing the supernatant, 1 mL of saponin solution was added and suspended, and 0.7 mL (sample) and 0.3 mL (negative control) were each dispensed into two tubes. After centrifuging again to remove the supernatant, 0.5 mL of saponin solution with anti-Troponin T-C (CT3) antibody added at 1:1000 as the primary antibody was added to the sample. 0.5 mL of saponin solution without antibody added was added to the negative control to suspend the cells, and the antibody was allowed to react overnight at 4°C. The next day, the primary antibody was removed, 0.5 mL of saponin solution with secondary antibody anti-Mouse IgG(H+L) Alexa Fluor 647 added at 1:500 was added, and it was shielded from light and allowed to react at room temperature for 1 hour. After 1 hour, the saponin solution with the secondary antibody added was replaced with 0.5 mL of PBS(-), and analysis was performed using FACS (Accuri TM CS6 Plus (manufactured by BD)).

[0125] <RNA Extraction and RT-qPCR> Troponin (a cardiomyocyte-specific gene), MYL2 (a ventricular muscle-specific gene which is one of the cardiomyocytes), and MYL7 (an atrial muscle-specific gene which is one of the cardiomyocytes) were used as the analysis targets.

[0126] (1) Reagents and Instruments RNA Extraction: miRNeasy Mini Kit (Product No. 217004, manufactured by Qiagen) cDNA Synthesis: ReverTra Ace(R) qPCR RT Master Mix with gDNA Remover (Product No. FSQ-301, manufactured by TOYOBO) qPCR Reaction: PowerUp SYBR Green Master Mix (Product No. A25776, manufactured by Thermo Fisher) QuantStudio 6 Flex Real-time PCR system (manufactured by Thermo Fisher) Nanophotometer Spectrophotometer C40 (manufactured by Wakem Biotech Co., Ltd.)

[0127] (2) RNA Extraction After removing the culture supernatants on the 12th and 19th days of induction of differentiation into cardiomyocytes, 0.5 mL of QIAZOL (manufactured by Qiagen) was added, suspended to lyse the cells, and the lysate was collected in a 1.5 ml tube. Thereafter, RNA was extracted according to the protocol attached to the miRNeasy Mini Kit, and the RNA concentration was measured with the Nanophotometer C40.

[0128] (3) RT-qPCR Using 1 μg of the RNA extracted above, a reverse transcription reaction was carried out according to the protocol attached to the PowerUp SYBR Green Master Mix to synthesize cDNA. Thereafter, a qPCR reaction was carried out by the standard method using 6 ng of cDNA. The calibration curve was prepared by collecting 10 μL each of 10 ng / μL of each cDNA sample, diluting it 1 / 10 therefrom, and making 5 points.

[0129] <Evaluation of Cardiomyocyte-Specific Gene Expression> Gene expression levels were analyzed using RNA extracted from cells on days 12 and 19 of differentiation induction using the QuantStudio 6 Flex Real-time PCR system. The primer sequences used are shown in Table 2, and the PCR conditions are shown in Table 3. Gene expression levels are shown as relative values, with the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) set at 1. The results are shown in Table 4.

[0130] <Evaluation of cell number> The cell clusters on days 12 and 19 of differentiation induction were dissociated into single cells, and then viable cells were counted (using a TC20 fully automated cell counter, Bio-Rad) using trypan blue staining (Bio-Rad). From the measured values ​​obtained above, the cell viability (%) (number of live cells at the time of measurement / total number of cells at the time of measurement × 100) and cell proliferation rate (%) (number of live cells at the time of measurement / number of cells at the time of cell seeding (start of cell culture) × 100) were calculated. The results are shown in Table 4.

[0131] <Evaluation of cardiac muscle purity> After dissociating the cell clusters on days 12 and 19 of differentiation induction into single cells, the cTnT positivity rate (number of cTnT positive cells / total number of cells constituting the cell cluster × 100) was calculated by FACS, and this was defined as myocardial purity (%). The results are shown in Table 4.

[0132] <Evaluation of heart rate> A video of the cells was taken on day 19 of differentiation induction, and the BPM (beats per minute, beats / min) of any spheroid was calculated. The results are shown in Table 4.

[0133] [Table 1]

[0134] [Table 2]

[0135] [Table 3]

[0136] [Table 4]

[0137] <Morphological observation> The formation of spheroids by each type of cell was observed using a phase-contrast microscope, and the results are shown in Figures 2 and 3.

[0138] <Result> On day 12 of differentiation induction, Examples 1 and 2 showed higher cell proliferation rates than Comparative Examples 1 and 2 due to the higher oxygen permeability of the culture bottom, particularly at a medium height of 10 mm. A similar trend was observed on day 19. Furthermore, Examples 1 and 2 showed higher myocardial purity than Comparative Examples 1 and 2 on days 12 and 19 of differentiation induction. Furthermore, when the expression of the myocardial-specific genes Troponin, MYL2, and MYL7 was compared, the expression levels of each gene were higher in Examples 1 and 2 than in Comparative Examples 1 and 2, particularly at a medium height of 10 mm. On day 19 of differentiation induction, Examples 1 and 2 showed a higher pulsation rate than Comparative Examples 1 and 2, and the pulsation rate was higher at a liquid depth of 10 mm than at a liquid depth of 5 mm. Furthermore, morphological observation revealed that Examples 1 and 2 formed multiple spheroids with uniform size and shape compared to Comparative Examples 1 and 2. Furthermore, Examples 1 and 2 demonstrated more efficient differentiation of iPS cells into cardiomyocytes than Comparative Examples 1 and 2.

[0139] [Example 3] Cultivation of human osteosarcoma-derived cancer cells (HOF-143B) Using the culture vessel prepared in Production Example 2, human osteosarcoma-derived cancer cells (HOF-143B) were cultured by the method described below to form spheroids.

[0140] Comparative Example 3 Culture was carried out in the same manner as in Example 3, except that a commercially available TCPS culture vessel (inner diameter of culture surface: 16 mm, 24-well plate, manufactured by Corning, made of polystyrene (PS)) with a culture surface thickness of 1000 μm was used.

[0141] Comparative Example 4 Culture was carried out in the same manner as in Example 3, except that a commercially available PDMS (polydimethylsiloxane) culture vessel with a culture member thickness of 350 μm (inner diameter of the culture surface: 15 mm, 24-well plate, product name G-plate, manufactured by VECELL, model number V24WGPB) was used as the highly oxygen-permeable vessel.

[0142] [Culture of human osteosarcoma-derived cancer cells (HOF-143B)] <Cell seeding> Culture medium was added to a centrifuge tube (50 ml) containing a cell suspension containing human osteosarcoma-derived cancer cells. The medium was prepared by adding 5 ml of fetal bovine serum (FBS, Fujifilm Wako Pure Chemical Industries), 0.5 ml of 200 mM L-glutamine solution (Fujifilm Wako Pure Chemical Industries), 0.05 ml of 1.5 mg / ml bromo-deoxy uridine (BUdR), 0.5 ml of non-essential amino acid (Fujifilm Wako Pure Chemical Industries), and 43.95 ml of E-MEM medium (containing 10 mg / ml phenol red and 2200 mg / ml sodium bicarbonate, culture grade, Fujifilm Wako Pure Chemical Industries). Cell density was adjusted by adjusting the cell number of the cell suspension containing human osteosarcoma-derived cancer cells. When 0.5 ml of cell suspension was seeded per well, the cell density was 1.0 x 10 4 cells / cm 2 The cell suspension was prepared so that

[0143] <Cell culture> A cell suspension containing human osteosarcoma-derived cancer cells was seeded onto the culture surface using a micropipette, then placed in an incubator and cultured at 37°C under 5% CO2. Culture was continued for 7 days. The medium was replaced on days 3 and 6 after cell seeding. The medium was replaced by leaving the culture vessel stationary, removing the supernatant once the spheroids had fully settled, and then adding the same amount of medium as had been removed.

[0144] <Morphological observation and evaluation> On day 7 of the culture, the cell morphology was observed using a phase contrast microscope and evaluated according to the following criteria. A: Spheroids are formed B: Cells adhere but do not form spheroids The observation results for Example 3 are shown in Figure 4. The evaluation results are shown in Table 5.

[0145] [Table 5]

[0146] <Result> In Comparative Examples 3 and 4, the cells adhered to the culture vessel and did not form spheroids, but in Example 3, the cells formed spheroids.

[0147] These results demonstrate that the use of the culture device of the present invention allows the formation of spheroids of cells that retain normal function. Furthermore, the use of the culture device of the present invention also allows for the efficient differentiation of iPS cells into cardiomyocytes. In other words, the culture device of the present invention facilitates the formation of spheroids and the differentiation of pluripotent stem cells. Therefore, the culture device of the present invention can be suitably used for drug discovery screening, diagnostics, and regenerative medicine.

Claims

1. A culture member for culturing cells, tissues, or organs on its culture surface, the culture member comprising a 4-methyl-1-pentene polymer (X), the culture surface having a water contact angle of more than 100° and not more than 160°, and an oxygen permeability of 4500 to 90000 cm at a temperature of 23°C and a humidity of 0%. ３ / (m ２ × 24h × atm), the cells, tissues, or organs are pluripotent stem cells or differentiated cells derived from pluripotent stem cells, The 4-methyl-1-pentene polymer (X) is contained in an amount of 90% by mass or more and less than 100% by mass in 100% by mass of the culture member; the 4-methyl-1-pentene polymer (X) is a copolymer (x1) of 4-methyl-1-pentene and at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene); When the amount of all repeating structural units of the 4-methyl-1-pentene polymer (X) is taken as 100 mol %, the content of structural units derived from 4-methyl-1-pentene is 80 to 98 mol %, and the content of structural units derived from at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is 2 to 20 mol %, the 4-methyl-1-pentene polymer (X) has a weight average molecular weight (Mw) of 30,000 to 500,000 and a molecular weight distribution (Mw / Mn) of 1.1 to 20; A culture member, wherein the culture surface is not subjected to processing to form an uneven structure.

2. The culture element described in claim 1, wherein the α-olefin is an α-olefin selected from the group consisting of propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, and 1-octadecene.

3. A culture device as described in claim 1, wherein the α-olefin is an α-olefin having 8 to 18 carbon atoms.

4. The culture member according to any one of claims 1 to 3, which is for forming spheroids.

5. The culture member according to any one of claims 1 to 4, wherein the cells, tissues, or organs are differentiated cells derived from iPS cells.

6. The culture member according to any one of claims 1 to 4, wherein the cells, tissues, or organs are cardiomyocytes derived from pluripotent stem cells.

7. The culture member according to any one of claims 1 to 6, wherein the culture member is in the form of a film or a sheet.

8. A culture device, at least a culture surface of which is formed from the culture member according to any one of claims 1 to 7.

9. A step (A) of contacting a cell, tissue, or organ with the culture surface of the culture member according to any one of claims 1 to 7 or the culture instrument according to claim 8; and A method for culturing cells, tissues, or organs, comprising the step (B) of culturing the cells, tissues, or organs in contact with the culture surface to form spheroids, A culture method, wherein the cells, tissues, or organs are pluripotent stem cells or differentiated cells derived from pluripotent stem cells.

10. The method for culturing cells, tissues, or organs according to claim 9, further comprising a step (C) of inducing differentiation of the cells, tissues, or organs, wherein step (C) is a step of inducing differentiation of iPS cells into cardiomyocytes by a protein-free cardiac differentiation induction (PFCD) method.

11. The method for culturing cells, tissues or organs according to claim 10, which increases myocardial purity.

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