JP2022154850A - Method for culturing cardiomyocytes - Google Patents

Abstract

To provide a method for culturing cardiomyocytes that can improve the functions of cardiomyocytes.SOLUTION: A method for culturing cardiomyocytes includes a step (A) for inoculating a culture face of a culture vessel with cardiomyocytes and a step (B) for culturing the cardiomyocytes in a medium. At least part of the culture face of the culture vessel is formed from a substrate comprising a 4-methyl-1-pentene polymer.SELECTED DRAWING: None

Description

The present invention relates to a method for culturing cardiomyocytes.

Since adult myocardial cells have poor self-renewal ability, it is extremely difficult to repair damaged myocardial tissue. In recent years, in order to repair damaged myocardial tissue, attempts have been made to transplant cardiomyocytes and/or tissue produced by in vitro culture into the affected area.
In addition, a method of differentiating human iPS cells to produce and culture cardiomyocytes has been developed, which is used in research to develop new therapeutic agents and treatment methods for heart disease and to elucidate the mechanisms that cause heart disease. ing. For example, Patent Literature 1 discloses a drug responsiveness test method using cardiomyocytes.

WO2019/131806

The present inventors have discovered a problem that when cardiomyocytes are cultured in vitro, the functions of cardiomyocytes may not be expressed appropriately depending on the culture conditions. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for culturing cardiomyocytes capable of improving the function of cardiomyocytes.

The present inventors have diligently studied to solve the above problems. As a result, the present inventors have found that the above problems can be solved by a culture method having the following configuration, and have completed the present invention. The present invention is, for example, the following [1] to [13].
[1] A method for culturing cardiomyocytes, comprising the step (A) of seeding cardiomyocytes on the culture surface of a culture vessel and the step (B) of culturing the cardiomyocytes in a medium, wherein the culture in the culture vessel A method for culturing cardiomyocytes, wherein at least part of the surface is formed from a substrate containing a 4-methyl-1-pentene polymer.
[2] the 4-methyl-1-pentene polymer is at least one selected from 4-methyl-1-pentene, ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene); The method for culturing cardiomyocytes according to [1], which is a copolymer of seeds.
[3] The method for culturing cardiomyocytes according to [1] or [2], wherein the entire culture surface of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer.
[4] The method for culturing cardiomyocytes according to any one of [1] to [3], wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells.
[5] The cardiomyocyte culture method of [4], wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells by a protein-free cardiomyocyte differentiation induction (PFCD) method.
[6] The cardiomyocyte culture method of [4] or [5], wherein the expression level of at least one gene selected from a cardiomyocyte marker and a sarcoplasmic reticulum marker is increased.
[7] the cardiomyocyte marker is at least one selected from cardiac troponin T (cardiac muscle) and MLC-2 (Myosin regulatory light chain 2, ventricular/cardiac muscle isoform); A method for culturing cardiomyocytes as described.
[8] The cardiomyocyte culture method of [6], wherein the sarcoplasmic reticulum marker is SERCA2 (sarcoplasmic/endoplasmic reticulum calcium ATPase 2).
[9] The method for culturing cardiomyocytes according to any one of [1] to [5], wherein the gene expression level of a factor involved in regulation of energy metabolism is increased.
[10] The factors involved in the regulation of energy metabolism include PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), PPARα (Peroxisome proliferator-activated receptor alpha), The method for culturing cardiomyocytes according to [9], which is at least one selected from glucose transporter members 4).
[11] The method for culturing cardiomyocytes according to any one of [1] to [5], wherein the expression level of an ischemic marker gene for cardiomyocytes is decreased.
[12] The cardiomyocyte culture method of [11], wherein the cardiomyocyte ischemia marker is BNP (brain natural peptide).
[13] The method for culturing cardiomyocytes according to any one of [1] to [12], wherein the maximum vertical distance from the culture surface of the culture vessel to the upper surface of the medium is greater than 7 mm.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for culturing cardiomyocytes capable of improving the function of cardiomyocytes.

FIG. 1 is a graph comparing the expression levels of cTnT. FIG. 2 is a graph comparing the expression levels of MYL2. FIG. 3 is a graph comparing the expression levels of ATP2A2. FIG. 4 is a graph comparing the expression levels of PPARGC1A. FIG. 5 is a graph comparing expression levels of PPARA. FIG. 6 is a graph comparing the expression levels of SLC2A4. FIG. 7 is a graph comparing the expression levels of BNP.

Next, the method for culturing cardiomyocytes of the present invention will be specifically described.
The present invention provides a method for culturing cardiomyocytes, comprising a step (A) of seeding cardiomyocytes in a culture vessel and a step (B) of culturing the cardiomyocytes in a medium, wherein the culture surface of the culture vessel is A method for culturing cardiomyocytes, at least a portion of which is formed from a substrate containing a 4-methyl-1-pentene polymer.
A culture vessel in which at least part of the culture surface is formed from a substrate containing a 4-methyl-1-pentene polymer is also referred to as a culture vessel (α).
In addition, the description “A to B” regarding the numerical range means that the range is A or more and B or less unless otherwise specified. For example, the description "1 to 5%" means 1% or more and 5% or less.
Also, in this specification, the term "step" is used not only for independent steps, but also when the intended purpose of the step is achieved even if it cannot be clearly distinguished from other steps. include.

[Culture vessel (α)]
At least part of the culture surface of the culture vessel (α) is formed from a substrate containing a 4-methyl-1-pentene polymer.

In the present invention, the culture vessel means all vessels used for culturing cells. As the culture vessel, various known culture vessels can be used, and the shape and size are not particularly limited. Examples of the culture vessel include dishes, flasks, plates, bottles, bags, tubes and the like. The culture vessels are typically used in devices such as incubators, mass culture devices, or perfusion culture devices.
Here, the culture surface means a surface with which the medium and/or the cells are in contact, or a surface to be contacted with the medium and/or the cells when the cells are cultured.

In the culture vessel, the phrase “at least a portion of the culture surface is formed from a substrate containing a 4-methyl-1-pentene polymer” means that at least a portion of the culture surface has a range of 4-methyl-1 -means formed from a substrate containing a pentene polymer, and the entire area of the culture surface may be formed from a substrate containing a 4-methyl-1-pentene polymer.

The culture vessel (α) is preferably a culture vessel whose bottom surface includes a culture surface in order to hold or store a culture medium. When the culture vessel (α) is, for example, a dish, flask or plate, the bottom surface includes the culture surface. It is formed from a substrate comprising a pentene polymer. When at least part or all of the bottom surface is formed from a substrate containing a 4-methyl-1-pentene polymer, oxygen is introduced into the medium through the substrate containing the 4-methyl-1-pentene polymer. can be efficiently supplied, making it easier to improve the function of cardiomyocytes in the medium. In addition, it becomes easier to culture cells at high density while maintaining the function of cardiomyocytes.

The culture vessel (α) preferably has a base material containing a 4-methyl-1-pentene polymer over the entire culture surface. That is, when the culture vessel (α) is, for example, a dish, a flask or a plate, the bottom includes the culture surface. It is preferably formed from a base material containing.

Although the thickness of the substrate containing the 4-methyl-1-pentene polymer is not particularly limited, it is preferably 20 μm to 400 μm, more preferably 20 μm to 300 μm, still more preferably 20 μm to 200 μm. The thickness of the base material containing the 4-methyl-1-pentene polymer is appropriately selected according to the form of the culture vessel. A moderate oxygen concentration in the medium can be easily obtained, and sufficient strength as a culture vessel can be easily obtained.

The culture vessel (α) is preferably a culture vessel having at least one well, more preferably a plate having at least one well, 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, A plate with wells such as 384 wells and 1536 wells is more preferable. In general, a culture vessel having a concave bottom surface such as a well needs to have a thick bottom surface in order to stabilize the complex shape of the bottom surface, which makes it difficult to sufficiently supply oxygen to cardiomyocytes. If the bottom is formed from a substrate containing a 4-methyl-1-pentene polymer, it has 1 well, 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells, etc. Even with a plate, the shape is stable and the oxygen supply to myocardial cells is sufficient.

The shape of the bottom surface of the culture vessel (α) is not particularly limited, and examples thereof include a flat bottom (F bottom), a round bottom (U bottom), a conical bottom (V bottom), a flat bottom with curved edges, and the like. When processing to a round bottom (U bottom), flat bottom (F bottom), conical bottom (V bottom), flat bottom + curved edge, etc., it may be processed at once by general injection molding or press molding, or a film Alternatively, it is also possible to prepare a sheet and perform secondary processing such as vacuum forming or pressure forming. The shape of the bottom surface is selected according to the purpose of culture, but when cardiomyocytes are cultured two-dimensionally, a flat bottom (F-bottom) is usually desirable, and when cardiomyocytes are cultured three-dimensionally, a round-bottom (U-bottom) is desirable. bottom) or a conical bottom (V-bottom) is usually desirable.

The portion of the culture vessel (α) other than the culture surface may be composed of a material other than the substrate containing the 4-methyl-1-pentene polymer. The material is not particularly limited, and known materials can be used. Such materials include, for example, polystyrene (PS), polydimethylsiloxane (PDMS), thermosetting resins, cyclic olefin polymers, cyclic olefin copolymers, glass, and the like.

The culture vessel (α) may be coated at least on its culture surface with a natural polymeric material, a synthetic polymeric material, or an inorganic material. Coating can be performed by a known method.

The coated culture vessel (α) has superior cardiomyocyte adhesion and proliferation. This is probably because the natural polymer material, synthetic polymer material, or inorganic material coated on the culture surface serves as a scaffold for myocardial cells. Therefore, when the cardiomyocytes are adhered and cultured, it is preferable to coat the culture vessel (α) with a natural polymer material, a synthetic polymer material, or an inorganic material before use.

The natural polymer material, synthetic polymer material, or inorganic material is not particularly limited, but examples of natural polymer materials include collagen, gelatin, alginic acid, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, fibronectin, laminin, fibrinogen, Osteopontin, tenascin, vitronectin, thrombospondin, agarose, elastin, keratin, chitosan, fibrin, fibroin, sugars, synthetic polymer materials such as polyglycolic acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, synthetic proteins , polyhydroxyethyl methacrylate, polyethyleneimine, and inorganic materials such as β-tricalcium phosphate and calcium carbonate.

Examples of the natural polymer material, synthetic polymer material, or inorganic material include vitrigel obtained by vitrifying a conventional hydrogel such as an extracellular matrix component and then rehydrating the vitrigel. For example, collagen vitrigel composed of a high-density collagen fiber network made from collagen, which is one of the extracellular matrix components, can be used.

From the viewpoint of improving cardiomyocyte adhesion and cardiomyocyte proliferation, and maintaining cardiomyocyte functions for a longer period of time, coating with proteins such as collagen, gelatin, laminin, polylysine, or peptides, or laminin is preferable. , collagen or polylysine is more preferred, and laminin coating is even more preferred. These coatings may be used singly or in combination of two or more.

At least the culture surface of the culture vessel (α) may be processed. Surface processing includes, for example, uneven structure forming processing, surface modification processing such as hydrophilic processing, and hydrophobic processing.

The method used for the surface modification treatment is not particularly limited. Vapor deposition, etching, addition of specific functional groups such as hydroxyl group, amino group, sulfone group, thiol group, carboxyl group, treatment with specific functional groups such as silane coupling, titanium coupling, zirconium coupling, oxidizing agent and physical treatment such as rubbing and sandblasting. These surface modification treatments may be performed singly or in combination of two or more. In addition, when surface modification treatment is performed, it is preferable to perform it at least on the culture surface.

At least the culture surface of the culture vessel (α) is preferably hydrophilized, more preferably corona-treated or plasma-treated. By hydrophilizing the surface of the culture vessel (α), the wettability of the surface of the culture vessel (α) is increased, the adhesion between the culture vessel (α) and the cardiomyocytes is improved, and the culture vessel (α) is improved. cardiomyocytes can grow uniformly on the surface of Further, by hydrophilizing the surface of the culture vessel (α), it becomes easier to coat the culture surface of the culture vessel (α) with a natural polymer material, a synthetic polymer material, or an inorganic material. In particular, the natural polymer material, synthetic polymer material, or inorganic material is uniformly loaded on the culture surface of the culture vessel (α), and it becomes easier to adhere. In the environment of cell culture, the natural polymer material, synthetic polymer material, or inorganic material does not peel off and can be used for cell culture while maintaining a stable initial state.
When plasma treatment is performed, nitrogen, hydrogen, helium, oxygen, argon, or the like is used as the accompanying gas, and at least one gas selected from nitrogen, helium, and argon is preferably selected.

The culture vessel (α) may be disinfected or sterilized to prevent contamination. The method of disinfection or sterilization is not particularly limited, and physical disinfection methods such as the circulation steam method, boiling method, intermittent method, and ultraviolet method, chemical disinfection methods using gases such as ozone, and disinfectants such as ethanol; Heat sterilization methods such as high pressure steam method and dry heat method; irradiation sterilization methods such as gamma ray method and high frequency method; gas sterilization methods such as ethylene oxide gas method and hydrogen peroxide gas plasma method. Among them, the ethanol sterilization method, the autoclave sterilization method, the gamma ray sterilization method, or the ethylene oxide gas sterilization method is preferable because the operation is simple and the sterilization can be performed sufficiently. These disinfection or sterilization treatments may be performed singly or in combination of two or more.

The culture surface of the culture vessel (α) preferably has a water contact angle of 50° to 100°, more preferably 55° to 100°, even more preferably 60° to 100°. Another preferred aspect of the water contact angle is 84° or less, more preferably 50° to 84°.

By adjusting the water contact angle of the culture surface of the culture vessel (α) within the above range, cardiomyocytes, for example, easily adhere to the culture surface and easily proliferate uniformly on the culture surface. In addition, the culture surface of the culture vessel (α) is evenly coated with a natural polymer material, a synthetic polymer material, or an inorganic material, making it easier to adhere. In a cell culture environment, the natural polymer material, synthetic polymer material, or inorganic material does not peel off and can be used for cell culture while maintaining a stable initial state.

The method for measuring the water contact angle is not particularly limited, and a known method can be used, preferably the sessile drop method. For the water contact angle, for example, the shape of a water droplet can be regarded as spherical under constant temperature and humidity conditions of 25 ± 5 ° C. and 50 ± 10% according to Japanese Industrial Standard JIS-R3257 (test method for wettability of substrate glass surface). A water droplet with a volume of 4 μL or less is dropped on the surface of a measurement sample made using the same material as the culture vessel, and the measurement sample is measured within 1 minute immediately after the water droplet contacts the measurement sample surface by the sessile drop method. It can be measured by a method of measuring the angle of the contact interface of water droplets.

The method of manufacturing the culture vessel (α) is not particularly limited, and the equipment used for manufacturing is also not limited. When the entire culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer, for example, a film or sheet containing the 4-methyl-1-pentene polymer is formed, and if necessary The film or sheet can be formed into a desired shape to produce a culture vessel. The culture vessel can also be obtained by direct molding by methods such as extrusion molding, solution casting molding, injection molding, and blow molding. When part of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer, for example, a film or sheet containing the 4-methyl-1-pentene polymer is formed, and the film or A culture vessel can be obtained by appropriately joining the sheet and other base material. The bonding method is not particularly limited, and the substrate containing the 4-methyl-1-pentene polymer and another substrate may be integrally formed, or they may be adhered to each other via an adhesive or pressure-sensitive adhesive. good.

As a method for forming the film or sheet, for example, a normal inflation method, a T-die extrusion method, or the like is employed. Manufacture is usually carried out warm. When the T-die extrusion method is employed, the extrusion temperature is preferably 100°C to 400°C, particularly preferably 200°C to 300°C. The roll temperature is preferably 45°C to 75°C, particularly preferably 55°C to 65°C.

Alternatively, the film or sheet may be produced by a solution casting method in which a 4-methyl-1-pentene polymer is dissolved in a solvent, poured onto a resin or metal, slowly dried while leveling, and formed into a film (sheet). good. The solvent used is not particularly limited, but hydrocarbon solvents such as cyclohexane, hexane, decane and toluene may be used. Two or more solvents may be mixed in consideration of the solubility and drying efficiency of the 4-methyl-1-pentene polymer. A polymer solution is applied by a method such as table coating, spin coating, dip coating, die coating, spray coating, bar coating, roll coating, or curtain flow coating, dried, and peeled off to form a film or sheet.

<4-methyl-1-pentene polymer>
In the present invention, 4-methyl-1-pentene homopolymers and copolymers of 4-methyl-1-pentene and other monomers are collectively referred to as "4-methyl-1-pentene polymers". .

Copolymers of 4-methyl-1-pentene, which is an example of 4-methyl-1-pentene polymers, with other monomers include random copolymers, alternating copolymers, block copolymers, and grafts. Any copolymer may be used. Copolymers of 4-methyl-1-pentene with other monomers include 4-methyl-1-pentene, ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene A copolymer with at least one olefin selected from ) is preferable because it has high strength, is resistant to breakage and cracking even when used as a base material, and has little deflection.

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

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. is mentioned. The olefin can be appropriately selected depending on the physical properties required for the base material. For example, the olefins are preferably α-olefins having 8 to 18 carbon atoms from the viewpoint of appropriate oxygen permeability and excellent rigidity, such as 1-octene, 1-decene, 1-dodecene, 1-tetradecene, At least one selected from 1-hexadecene, 1-heptadecene and 1-octadecene is more preferred. When the number of carbon atoms in the olefin is within the above range, the processability of the polymer is improved, and the appearance of the base material tends to be less likely to occur due to cracks or cracks at the edges. In addition, the defective product rate of the base material is reduced.

The olefin can be used alone or in combination of two or more. From the viewpoint of the strength of the material, the number of carbon atoms is preferably 2 or more, 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.

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 99.5 mol%, still more preferably 85 to 98 mol %.
Further, the 4-methyl-1-pentene polymer is at least one selected from 4-methyl-1-pentene, ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) If it is a copolymer with an olefin, it is derived from at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer The content of the structural unit is preferably 0 to 40 mol%, more preferably 0.5 to 20 mol%, still more preferably 2 to 15 mol%. The content of these structural units is based on 100 mol % of all repeating structural units in the 4-methyl-1-pentene polymer. When the content of the structural unit is within the above range, a uniform culture surface with excellent workability can be obtained, and the toughness and strength of the film are well-balanced, so that deflection is reduced.

The 4-methyl-1-pentene polymer is derived from structural units derived from 4-methyl-1-pentene and the ethylene and an α-olefin having 3 to 20 carbon atoms within a range that does not impair the effects of the present invention. It may have structural units other than structural units (hereinafter also referred to as "other structural units"). The content of other structural units is, for example, 0 to 10.0 mol %. When the 4-methyl-1-pentene polymer has other structural units, the other structural units may be one or two or more.

Examples of monomers leading to other structural units include cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins, and halogenated olefins. Cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins and halogenated olefins include, for example, paragraphs [0035] to [0041] of JP-A-2013-169685. compounds can be used.

The 4-methyl-1-pentene polymer may be used singly or in combination of two or more.

A commercial product can also be used as the 4-methyl-1-pentene polymer. Specific examples include TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31 and DX845 manufactured by Mitsui Chemicals, Inc.). Also, 4-methyl-1-pentene polymers manufactured by other manufacturers can be preferably used as long as they satisfy the above requirements. These commercial products may be used singly or in combination of two or more.

A 4-methyl-1-pentene polymer usually has a melting point of 200° C. to 240° C. and high heat resistance. Further, since it does not undergo hydrolysis and has excellent water resistance, boiling water resistance and steam resistance, a substrate containing a 4-methyl-1-pentene polymer can be subjected to autoclave sterilization. The 4-methyl-1-pentene polymer also has a high visible light transmittance (usually 90% or more) and does not emit autofluorescence, so it is formed from a base material containing a 4-methyl-1-pentene polymer. The culture vessel is designed to facilitate observation of cultured cells. Furthermore, since it exhibits excellent chemical resistance to most chemicals and does not easily sorb drugs, it does not interfere with the effects of drugs for maintaining cardiomyocytes, and is also suitable for drug discovery screening and diagnostic applications. be done. The 4-methyl-1-pentene polymer can be heat-sealed, and can be easily heat-sealed not only with itself but also with other materials. In addition, since it can be thermoformed, it can be easily formed into a culture vessel of any shape, for example, it can be easily formed using an imprint method or an insert method.

The weight average molecular weight (Mw) of the 4-methyl-1-pentene polymer, measured by gel permeation chromatography (GPC) using standard polystyrene as a reference material, is preferably 10,000 to 2,000,000, more preferably 20,000. ~1,000,000, more preferably 30,000 to 500,000. Here, the sample concentration for GPC measurement can be, for example, 1.0 to 5.0 mg/ml. Further, the molecular weight distribution (Mw/Mn) of the 4-methyl-1-pentene polymer is preferably 1.0-30, more preferably 1.1-25, still more preferably 1.1-20. The solvent used in GPC is preferably ortho-dichlorobenzene. In addition, as an example of the measurement conditions, the conditions shown in Examples to be described later can be cited, but the measurement conditions are not limited to these.

By setting the weight average molecular weight (Mw) to the above upper limit or less, the film produced by melt molding in the molding method of the 4-methyl-1-pentene polymer described later can easily suppress the occurrence of defects such as gel. It becomes easier to form a film with a uniform surface. In addition, when the film is produced by the solution casting method, the solubility in the solvent is improved, so that problems such as film gel can be easily suppressed, making it easier to form a film with a uniform surface.

Further, by setting the weight-average molecular weight (Mw) to the lower limit or more, the strength of the culture vessel tends to be sufficient. Furthermore, by setting the molecular weight distribution within the above range, it is easy to suppress the stickiness of the surface of the culture vessel produced, and the toughness of the culture vessel tends to be sufficient, so bending during molding and cracking during cutting are prevented. easier to suppress.

The weight-average molecular weight (Mw) and molecular weight distribution (Mw/Mn) of the 4-methyl-1-pentene polymer are different from each other when two or more types are used as the 4-methyl-1-pentene polymer. , Mw and Mw/Mn should be within the above ranges.

The 4-methyl-1-pentene polymer preferably has an oxygen permeability coefficient of 100 to 2500 cm ³ ×mm/(m ² ×24h×atm), more preferably 1000 to 2500 cm ³ ×mm/(m ² ×24h×atm). ) is more preferable. When the oxygen permeability coefficient is within the above range, the oxygen permeability is excellent, so that the cardiomyocytes maintain good morphology and are likely to proliferate efficiently according to the culture period.

Specifically, the oxygen permeability coefficient can be measured by the following method. A measurement sample was prepared from a film composed of a 4-methyl-1-pentene polymer, and the oxygen permeability coefficient [cm ³ × mm/(m ² x 24 h x atm)]. The instrument used for the measurement is not particularly limited as long as it uses a differential pressure type gas permeability measurement method. A measurement sample was prepared by cutting a 90×90 mm test piece from a 50 μm-thick film composed of a 4-methyl-1-pentene polymer, and the diameter of the measurement part was 70 mm (the transmission area was 38.46 cm ² ). It is preferable to do so. Since the oxygen permeability is high, it is more preferable to apply an aluminum mask to the sample in advance so that the actual permeation area is 5.0 cm ² . The sample to be measured may or may not have been subjected to microfabrication or surface modification treatment, but it is preferable that the sample has not been subjected to any treatment. A value obtained by dividing the oxygen permeability coefficient by the thickness (μm) of the base material is defined as the oxygen permeability [cm ³ /(m ² ×24h×atm)].

Since the 4-methyl-1-pentene polymer has such excellent properties as described above, a culture vessel having at least a culture surface formed of a base material containing the 4-methyl-1-pentene polymer is suitable for culture. It has good shape stability, light transmittance, molding processability, and oxygen permeability, and can be sterilized. Are better.

<Method for producing 4-methyl-1-pentene polymer>
The method for producing the 4-methyl-1-pentene polymer may be any method as long as it can polymerize 4-methyl-1-pentene, olefins and other monomers. Also, a chain transfer agent such as hydrogen may coexist in order to control the molecular weight and molecular weight distribution. The equipment used for manufacturing is also 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. A slurry method and a solution method are preferred. Further, 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 a plurality of polymers having different molecular weights are blended in a polymerization system. When hydrogen is used as a chain transfer agent in either single-stage or multi-stage polymerization, it may be charged all at once or dividedly, for example, at the beginning, middle and end of the polymerization. The polymerization may be carried out at normal temperature, or may be heated if necessary, but from the viewpoint of polymerization efficiency, it is preferably carried out at 20°C to 80°C, and particularly preferably at 40°C to 60°C. . The catalyst used for production is also not limited, but from the viewpoint of polymerization efficiency, it is preferable to use, for example, the solid titanium catalyst component (I) described in WO 2006/054613.

Incidentally, when the substrate containing the 4-methyl-1-pentene polymer is a composition containing the 4-methyl-1-pentene polymer, 4-methyl-1- The pentene polymer is preferably 90% by mass or more and less than 100% by mass, more preferably 95% by mass or more and less than 100% by mass, and particularly preferably 99% by mass or more and less than 100% by mass. When a large amount of components other than the 4-methyl-1-pentene polymer is contained, not only the oxygen permeability is lowered, but also the transparency and strength are lowered.
Components other than the 4-methyl-1-pentene polymer include heat stabilizers, light stabilizers, processing aids, plasticizers, antioxidants, lubricants, antifoaming agents, antiblocking agents, colorants, modifiers, Additives such as pesticides, antibacterial agents, antifungal agents, and antifogging agents are included.

[Cardiomyocytes]
Cardiomyocytes may be cardiomyocytes differentiated from pluripotent stem cells, or primary cultured cardiomyocytes isolated from the heart of an organism. In addition, commercially available cardiomyocytes derived from pluripotent stem cells, for example, iCell Cardiomyocytes from FUJIFILM Cellular Dynamics, MiraCell Cardiomyocytes v2 from Takara Bio, Cor. 4U, Myoridge's CarmyA, REPROCELL's ReproCardio2, and the like may be used.

Pluripotent stem cells are a general term for stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Pluripotent stem cells include, for example, embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), trophoblast stem cells (TS cells), epiblast stem cells ( epiblast stem cells (EpiS cells), embryonic germ cells (EG cells), multipotent germline stem cells (mGS cells), induced pluripotent stem cells (iPS cells) , Muse cells (Multi-lineage differentiating Stress Enduring cells) and the like. Pluripotent stem cells are preferably ES cells or iPS cells.

Cardiomyocytes are preferably cardiomyocytes differentiated from pluripotent stem cells, more preferably cardiomyocytes differentiated from induced pluripotent stem cells. Cardiomyocytes differentiated from induced pluripotent stem cells can be prepared by a known cardiomyocyte differentiation induction method, for example, a protein-free cardiomyocyte differentiation induction (PFCD) method can be used (WO 2015/182765). ).
Cardiomyocytes are preferably cardiomyocytes differentiated from induced pluripotent stem cells by a protein-free cardiomyocyte differentiation induction (PFCD) method.

Cardiomyocytes are not particularly limited in origin, and may be derived from mammals, birds, amphibians, reptiles, fish, etc., preferably mammals, more preferably humans, monkeys, mice, rats, pigs, It is derived from dogs, sheep, cats, and goats, more preferably from humans.
Cardiomyocytes are preferably cardiomyocytes differentiated from human-derived embryonic stem cells or cardiomyocytes differentiated from human-derived induced pluripotent stem cells.

Cardiomyocytes may be normal cardiomyocytes, cardiomyocytes containing genetic mutations, or disease model cardiomyocytes.
In the culture vessel (α), since oxygen can be efficiently supplied to the medium through the substrate containing the 4-methyl-1-pentene polymer, both adherent cardiomyocytes and floating cells can be cultured. can also be suitably cultured, but cardiomyocytes are preferably adherent.

[Step (A)]
Step (A) is a step of seeding cardiomyocytes on the culture surface of the culture vessel (α). The method of seeding the cardiomyocytes on the culture surface of the culture vessel (α) is not particularly limited. After the cardiomyocytes are evenly dispersed in the culture container by moving the culture container, the culture container is allowed to stand still in the incubator.

The cardiomyocyte seeding density is not particularly limited as long as the cardiomyocytes can be maintained or proliferated, but is preferably 0.1×10 ⁵ cells/cm ² to 10.0×10 ⁵ cells/cm ² , More preferably 0.3×10 ⁵ cells/cm ² to 5.0×10 ⁵ cells/cm ² , still more preferably 0.5×10 ⁵ cells/cm ² to 3.0×10 ⁵ cells/cm ² .
When the cardiomyocyte seeding density is within the above range, the cardiomyocytes proliferate more efficiently than when the cardiomyocyte seeding density is outside the above range, which is preferable.

The medium used for seeding is not particularly limited as long as it allows myocardial cells to survive, and may be appropriately selected according to the cell type used. The medium used for seeding may be the medium used in step (B) described below. The medium used for seeding is, for example, any cell culture basal medium, differentiation medium, primary culture medium, etc. Specifically, Eagle's small essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham F-12, MCDB medium, Williams medium E, Seeding medium for CarmyA (manufactured by Myoridge), Cardiac Myocyte Medium (manufactured by ScienceCell Research Laboratories), iCell Cardiomyocytes Plating Medium (manufactured by ScienceCell Research Laboratories) Dynamics), mixed media thereof, and the like. Furthermore, a medium supplemented with serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, etc. may be used.

The amount of medium used for seeding is not particularly limited, but when added to the culture vessel, the longest distance in the vertical direction from the culture surface of the culture vessel to the top surface of the medium is preferably greater than 7 mm, and greater than 7 mm and 20 mm. It is more preferably less than or equal to, and more preferably greater than 7 mm and 15 mm or less.
Cultivation temperature is also not particularly limited, but is usually carried out at about 25 to 40°C.

[Step (B)]
Step (B) is a step of culturing the cardiomyocytes seeded in step (A) in a medium. A method for culturing cardiomyocytes in a medium is not particularly limited, and may be carried out according to a known protocol, and commercially available medium and culture kits may be used.

The medium used for culture is not particularly limited as long as it is a medium in which cardiomyocytes can proliferate, and may be appropriately selected according to the cell type used. The medium used for culture is, for example, any cell culture basal medium, differentiation medium, primary culture medium, etc. Specifically, Eagle's small essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham F-12, MCDB medium, Williams medium E, CarmyA maintenance medium (manufactured by Myoridge), CardioGro (manufactured by Funakoshi), Cardiac Myocyte Medium (manufactured by ScienceCell Research Laboratories), iCell Cardiomyocytes Maintenance Medium (manufactured by FUJIFILM Cellular Dynamics), mixed media thereof, and the like. Furthermore, a medium supplemented with serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, etc. may be used.

The amount of medium used for culture is not particularly limited, but when added to the culture vessel, the longest distance in the vertical direction from the culture surface of the culture vessel to the top surface of the medium is preferably greater than 7 mm, and greater than 7 mm and 20 mm. It is more preferably less than or equal to, and more preferably greater than 7 mm and 15 mm or less.
The frequency of medium replacement is not particularly limited, but it is preferably performed every day.
Although the culture temperature is not particularly limited, it is usually carried out at about 25 to 40°C.

The culture period is not particularly limited, and the culture may be continued until the myocardial cells proliferate sufficiently in the culture vessel and exhibit their functions. The culture period is preferably 1 to 21 days, more preferably 3 to 14 days, still more preferably 5 to 10 days.

In the culture vessel (α), since oxygen can be efficiently supplied to the medium through the base material containing the 4-methyl-1-pentene polymer, both adherent culture and suspension culture can be suitably performed. Adherent culture is preferred.

Step (B) is performed after step (A) or simultaneously with step (A).

The method for culturing cardiomyocytes of the present invention may include step (I) of pre-culturing cardiomyocytes prior to step (A).
The method for precultivating cardiomyocytes is not particularly limited, and may be performed according to known protocols, and commercially available media and culture kits may be used.

The medium used for pre-culture is not particularly limited as long as it is a medium in which cardiomyocytes can proliferate, and may be appropriately selected according to the cell type used. The medium used for pre-culture is, for example, any cell culture basal medium, differentiation medium, primary culture medium, etc. Specifically, Eagle's small essential medium (EMEM), Dulbecco's modified Eagle medium (DMEM), Glasgow MEM (GMEM), IMDM, RPMI1640, Ham F-12, MCDB medium, Williams medium E, CarmyA maintenance medium (manufactured by Myoridge), CardioGro (manufactured by Funakoshi), Cardiac Myocyte Medium (manufactured by ScienceCell Research Laboratories), Examples thereof include iCell Cardiomyocytes Maintenance Medium (manufactured by FUJIFILM Cellular Dynamics) and mixed media thereof. Furthermore, a medium supplemented with serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, etc. may be used.

The amount of medium used for preculture is not particularly limited.
The frequency of medium exchange in pre-culture is not particularly limited, but it is preferably carried out every day.
Although the culture temperature in the pre-culture is not particularly limited, it is usually carried out at about 25 to 40°C.

The pre-culture period is not particularly limited, and the culture may be performed until cardiomyocytes proliferate sufficiently to obtain the desired number of cells. The culture period is preferably 1 to 10 days, more preferably 2 to 7 days, still more preferably 3 to 6 days.

[Cardiomyocyte function]
In the method for culturing cardiomyocytes of the present invention, the function of cardiomyocytes can be improved by culturing the cardiomyocytes in the culture vessel (α). Cardiomyocyte function can be evaluated by expression levels of genes for cardiomyocyte markers, sarcoplasmic reticulum markers, factors involved in energy metabolism control, cardiomyocyte ischemia markers, and the like.

The cardiomyocyte culture method of the present invention preferably increases the expression level of at least one gene selected from cardiomyocyte markers and sarcoplasmic reticulum markers.

The above-mentioned increase refers to the increase in cardiomyocyte culture under the same experimental conditions as the cardiomyocyte culture method of the present invention, using a polystyrene cell culture vessel instead of the culture vessel (α). , means that the expression level of at least one gene selected from cardiomyocyte markers and sarcoplasmic reticulum markers is used as a control, and the expression level is higher than that of the control. The expression level is preferably 2 times or more higher than that of the comparative object, and more preferably 3 times or more.
Gene expression levels can be measured by known methods, such as quantitative RT-PCR.

The cardiomyocyte marker is not particularly limited and includes, for example, Troponin C (slow skeleton and cardiac muscles), Troponin I (cardiac muscle), Troponin T (cardiac muscle), Myosin regulatory light ｃｈａｉｎ ２，ｖｅｎｔｒｉｃｕｌａｒ／ｃａｒｄｉａｃ ｍｕｓｃｌｅ ｉｓｏｆｏｒｍ（ＭＬＣ－２、Ｍｙｏｓｉｎ Ｌｉｇｈｔ Ｃｈａｉｎ ２）、Ｍｙｏｓｉｎ ｒｅｇｕｌａｔｏｒｙ ｌｉｇｈｔ ｃｈａｉｎ ７（ＭＬＣ－２ａ、Ｍｙｏｓｉｎ ｒｅｇｕｌａｔｏｒｙ ｌｉｇｈｔ ｃｈａｉｎ ２，ａｔｒｉａｌ ｉｓｏｆｏｒｍ）、Ｈｏｍｅｏｂｏｘ ｐｒｏｔｅｉｎ Ｎｋｘ－２．５、Ｐｏｔａｓｓｉｕｍ ｖｏｌｔａｇｅ－ ｇａｔｅｄ ｃｈａｎｎｅｌ ｓｕｂｆａｍｉｌｙ ＫＱＴ ｍｅｍｂｅｒ １、Ｖｏｌｔａｇｅ－ｄｅｐｅｎｄｅｎｔ Ｌ－ｔｙｐｅ ｃａｌｃｉｕｍ ｃｈａｎｎｅｌ ｓｕｂｕｎｉｔ ａｌｐｈａ－１Ｃ、Ｓｏｄｉｕｍ ｃｈａｎｎｅｌ ｐｒｏｔｅｉｎ ｔｙｐｅ ５ ｓｕｂｕｎｉｔ ａｌｐｈａ、Ｐｏｔａｓｓｉｕｍ ｖｏｌｔａｇｅ－ｇａｔｅｄ ｃｈａｎｎｅｌ ｓｕｂｆａｍｉｌｙ Ｈ ｍｅｍｂｅｒ ２（ｈＥＲＧ１）等が挙げられる。 The cardiomyocyte marker is preferably at least one selected from troponin T (cardiac muscle), myosin regulatory light chain 2, and ventricular/cardiac muscle isoform (MLC-2).

Human genes encoding the cardiomyocyte markers (proteins) are shown in Table 1. Therefore, in the cardiomyocyte culture method of the present invention, when the cardiomyocytes are human-derived, it is preferable that the expression level of at least one human gene selected from the human genes listed in Table 1 is increased. It is preferable that the expression level of at least one selected from TNNT2) and MYL2 is increased.

The sarcoplasmic reticulum marker is not particularly limited, and is preferably sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).

Human genes encoding the sarcoplasmic reticulum markers (proteins) are shown in Table 2. Therefore, in the cardiomyocyte culture method of the present invention, when the cardiomyocytes are human-derived, it is preferable that the expression level of at least one human gene selected from the human genes listed in Table 2 is increased. It is more preferable that the expression level is increased.

The method for culturing cardiomyocytes of the present invention preferably increases the expression level of genes of factors involved in energy metabolism control.

The above-mentioned increase refers to the increase in cardiomyocyte culture under the same experimental conditions as the cardiomyocyte culture method of the present invention, using a polystyrene cell culture vessel instead of the culture vessel (α). , means that the expression level of the gene of the factor involved in the control of energy metabolism is higher than that of the comparison control. The expression level is preferably 2 times or more higher than that of the comparative object, and more preferably 3 times or more.
Gene expression levels can be measured by known methods, such as quantitative RT-PCR.

As used herein, the term "energy metabolism" is used as a generic term for chemical reactions for supplying energy to cells, and includes sugar metabolism, lipid metabolism, and the like.
The factor involved in energy metabolism control is not particularly limited as long as it is a factor involved in enhancement or suppression of energy metabolism, but preferably a factor involved in enhancement of energy metabolism.

Since the heart requires a lot of energy to continuously pump blood throughout the body, normal myocardial cells expend a large amount of energy to maintain membrane potential, reuptake Ca ions, and contract with myosin. be. To meet its high energy demand, it produces and consumes large amounts of ATP. 70 to 90% of the ATP produced is derived from fatty acid oxidation, and 10% to 30% is derived from sugar and lactic acid oxidation, ketone bodies and amino acid oxidation.
Fatty acids are taken up via CD36 and Fatty-Acid Binding Protein (FABP) present in the cell membrane of myocardial cells, and are bound to FABP or converted into Acyl-CoA in cells. Fatty acids taken into cells are taken into mitochondria to become fatty acid Acyl-CoA, which is β-oxidized and used for ATP production.
Glucose is taken up into cells by sugar transporters called GLUTs. Glucose taken up into cells is metabolized into pyruvate through glycolysis, and under anaerobic conditions, anaerobic glycolysis is decomposed into lactate. used for production.

The factor involved in the control of energy metabolism is preferably a factor involved in the enhancement of ATP production, more preferably a factor involved in the enhancement of ATP production from fatty acids or glucose, uptake or β-oxidation of fatty acids or glucose. is more preferably a factor that promotes

Factors involved in energy metabolism control are preferably transporters of energy metabolism substrates, enzymes involved in the ATP production system (β-oxidation, glycolysis, anaerobic glycolysis, TCA cycle or oxidative phosphorylation), or these is a transcription factor or transcription cofactor that regulates the expression of

The factor involved in the regulation of energy metabolism is preferably selected from PGC-1 (Peroxisome proliferator-activated receptor gamma coactivator 1), PPAR (Peroxisome proliferator-activated receptor), and GLUT (Solute carrier family 2, facilitator)少なくとも１種であり、より好ましくはＰＧＣ－１α（Ｐｅｒｏｘｉｓｏｍｅ ｐｒｏｌｉｆｅｒａｔｏｒ－ａｃｔｉｖａｔｅｄ ｒｅｃｅｐｔｏｒ ｇａｍｍａ ｃｏａｃｔｉｖａｔｏｒ １－ａｌｐｈａ）、ＰＰＡＲα（Ｐｅｒｏｘｉｓｏｍｅ ｐｒｏｌｉｆｅｒａｔｏｒ－ａｃｔｉｖａｔｅｄ ｒｅｃｅｐｔｏｒ ａｌｐｈａ）、及びＧＬＵＴ－４（Ｓｏｌｕｔｅ ｃａｒｒｉｅｒ ｆａｍｉｌｙ ２，ｆａｃｉｌｉｔａｔｅｄ ｇｌｕｃｏｓｅ ｔｒａｎｓｐｏｒｔｅｒ ｍｅｍｂｅｒ ４ ) is at least one selected from

Peroxisome proliferator-activated receptor (PPAR) is a transcription factor belonging to the NR1C family of nuclear receptors, and three isoforms of PPARα, PPARβ/δ and PPARγ have been reported. PPARs regulate the transcription of genes involved in fatty acid metabolism and the like, and particularly in cardiomyocytes, mainly promote β-oxidation and fatty acid uptake.
PPAR is preferably PPARα or PPARβ/δ, more preferably PPARα, because it is highly expressed in myocardial cells and greatly involved in ATP production enhancement.

Human genes encoding the PPARs (proteins) are shown in Table 3. Therefore, in the cardiomyocyte culture method of the present invention, when the cardiomyocytes are human-derived, it is preferable that the expression level of at least one human gene selected from the human genes listed in Table 3 is increased. It is more preferable that the expression level of at least one selected from PPARD is increased, and it is further preferable that the expression level of PPARA is increased.

A sugar transporter (Solute carrier family 2, facilitated glucose transporter) is a transporter that performs facilitated diffusion-type transport of sugar (mainly glucose), and increases the intracellular concentration of sugar, which is a metabolic substrate.
The sugar transporter is, for example, GLUT-1 (Solute carrier family 2, facilitated glucose transporter member 1), GLUT-2, GLUT-3, GLUT-4, GLUT-5, GLUT-6, GLUT-7, GLUT-8 , GLUT-9, GLUT-10, GLUT-11, GLUT-12, Hmit (Proton myo-inositol cotransporter), GLUT-14 and the like.
The sugar transporter is preferably GLUT-1 or GLUT-4, more preferably GLUT-4, since it is highly expressed in myocardial cells and greatly involved in the enhancement of ATP production.

Human genes encoding the sugar transporters (proteins) are shown in Table 4. Therefore, in the cardiomyocyte culture method of the present invention, when the cardiomyocytes are human-derived, it is preferable that the expression level of at least one selected from the human genes listed in Table 4 is increased, and SLC2A1 ( It is more preferable that the expression level of at least one selected from GLUT1) and SLC2A4 (GLUT4) is increased, and it is further preferable that the expression level of SLC2A4 (GLUT4) is increased.

PGC-1 (PPARγ coactivator 1) is a protein identified as a transcriptional coactivator that binds to PPARγ and enhances its transcriptional activity, and isoforms of PGC-1α and PGC-1β have been reported. PGC-1 regulates the expression of multiple genes involved in mitochondrial biogenesis and ATP production. PGC-1α has a function of promoting mitochondrial synthesis, and also increases GLUT4. In addition to PPARγ, PGC-1α also acts as a coactivator of various nuclear receptors such as PPARα, PPARδ, TRα (Thyroid Receptor α), and ERα (Estrogen Receptor α). Acts as a coactivator of ERR (Estrogen Receptor-related Receptor).
PGC-1 is preferably PGC-1α because it is greatly involved in the enhancement of ATP production.

Human genes encoding the PGC-1 (protein) are shown in Table 5. Therefore, in the cardiomyocyte culture method of the present invention, when the cardiomyocytes are human-derived, it is preferable that the expression level of at least one human gene selected from the human genes listed in Table 5 is increased. It is more preferable that the expression level is increased.

The cardiomyocyte culture method of the present invention preferably reduces the expression level of the cardiomyocyte ischemia marker gene.

The aforementioned decrease refers to the number of cardiomyocytes cultured under the same experimental conditions as the cardiomyocyte culture method of the present invention, except that polystyrene cell culture vessels were used instead of the culture vessel (α). , means that the expression level of an ischemic marker gene for myocardial cells is lower than that of a control, which is used as a control. The expression level is preferably 2 times or more lower than that of the comparative object, and more preferably 3 times or more.
Gene expression levels can be measured by known methods, such as quantitative RT-PCR.

The cardiomyocyte ischemia marker is not particularly limited, and examples thereof include BNP (brain natriuretic peptide) and NT-proBNP (N-terminal proBNP) produced by cleavage of its precursor, proBNP. The cardiomyocyte ischemia marker is preferably BNP.

Table 6 shows the human genes encoding the cardiomyocyte ischemia markers (proteins). Therefore, in the cardiomyocyte culture method of the present invention, when the cardiomyocytes are human-derived, it is preferable that the expression level of at least one human gene selected from the human genes listed in Table 6 is reduced, preferably The expression level of BNP is decreased.

The cardiomyocyte culture method of the present invention is preferably used to promote cardiomyocyte maturation, and more preferably to increase the expression level of at least one gene selected from cardiomyocyte markers and sarcoplasmic reticulum markers. used for
The method for culturing cardiomyocytes of the present invention is preferably used to enhance the energy metabolism of cardiomyocytes, and more preferably to increase expression levels of genes of factors involved in regulation of energy metabolism.
The cardiomyocyte culture method of the present invention is preferably used to improve the oxygen condition of cardiomyocytes, and more preferably to decrease the expression level of ischemic marker genes in cardiomyocytes.

The method for culturing cardiomyocytes of the present invention can be used for drug discovery, such as screening drugs for heart disease and evaluating drug effects, cardiotoxicity, responsiveness, etc., and for research purposes for elucidating the mechanisms that cause heart disease. can be used in In addition, the method for culturing cardiomyocytes of the present invention can be used to produce cardiomyocytes for transplantation into diseased areas.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these.

[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 Examples were measured by gel permeation chromatography (GPC).
Specifically, under the following conditions, the weight average molecular weight (Mw) and number average molecular weight (Mn) of the polymer dissolved in ortho-dichlorobenzene were measured by calibrating the molecular weight with standard polystyrene.
・Apparatus: Gel permeation chromatograph HLC-8321 GPC/HT type (manufactured by Tosoh Corporation)
・ Data analysis software: Empower3 (manufactured by Waters)
・Detector: Differential refractometer ・Series connection column: TSKgel GMH6-HT (2) and TSKgel GMH6-HTL (2)
・Column temperature: 140°C
・Flow rate: 1.0 ml/min ・Sample concentration: 1.5 mg/ml

[Production Example 1] Production of base material TPX (registered trademark) which is a 4-methyl-1-pentene polymer (manufactured by Mitsui Chemicals, Inc.: molecular weight (Mw) = 428000, molecular weight distribution (Mw/Mn) = 4.1 ), put it into an extruder with a T-die equipped with a full-flight screw for extruding the base layer, set the extrusion temperature to 270 ° C., the roll temperature to 60 ° C., and change the roll rotation speed conditions. A film 1 having a thickness of 50 μm was obtained by extrusion.

Using the film 1 as a measurement sample, the oxygen permeability coefficient [cm ³ × mm/(m ² × 24 h x atm)] was measured. The diameter of the measuring portion was 70 mm (transmission area was 38.46 cm ² ). Since the oxygen permeability coefficient was expected to be large, the sample was masked with aluminum in advance to set the actual permeation area to 5.0 cm ² . The oxygen permeability coefficient was 1912 cm ³ ×mm/(m ² ×24h×atm).

The film 1 was plasma-treated using an atmospheric pressure plasma surface treatment apparatus (manufactured by Sekisui Chemical Co., Ltd.) in which the chamber was filled with a stream of nitrogen (treatment speed: 2 m/min, output: 4.5 kW, 2 reciprocations).

Using the plasma-treated film 1 as a measurement sample, the water contact angle was measured. The water contact angle was measured according to Japanese Industrial Standards JIS-R3257 (testing method for wettability of substrate glass surface). Under constant temperature and humidity conditions of 25 ± 5 ° C and 50 ± 10%, a water droplet of a volume of 4 μL or less that can be regarded as a spherical water droplet is dropped on the surface of the measurement sample. The angle of the contact interface between the measurement sample and the water droplet was measured within 1 minute from immediately after the contact. The water contact angle of plasma-treated Film 1 was 60.3°.

[Production Example 2] Preparation of culture vessel The plasma-treated film 1 was cut into a size of 8 cm × 12 cm, and attached to the bottom of a 24-well container frame made of polystyrene (also referred to as PS) via a medical adhesive (manufactured by 3M). A 24-well culture plate was prepared. Then, it was packed in a gamma-ray resistant bag and irradiated with 10 kGy of gamma rays for sterilization. This was designated as a T plate. The culture area of one well was approximately 2 cm ² .

[Example 1] Cultivation of cardiomyocytes Using the T plate prepared in Production Example 2, cardiomyocytes were cultured by the method described later. The medium was used so that the medium depth was 2 mm (medium volume: 0.35 mL).

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

[Comparative Example 1]
In the same manner as in Example 1, except that a PS culture vessel (CellBIND ^™ , manufactured by Corning, culture area per well is about 2 cm ² , 24-well plate, hereinafter also referred to as PS plate or Corning) was used. cultured.

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

[Cardiomyocyte culture]
<Preparation of media and reagents>
Human iPS cell-derived cardiomyocytes (manufactured by Myoridge Inc.) cryopreserved in liquid nitrogen were used.
The reagents used are as follows. All were refrigerated.
・ Maintenance medium for CarmyA (product code ME-01, manufactured by Myoridge, lot number P-K05V4-3, P-A15V4-3)
・ Seeding medium for CarmyA (product code ME-02, manufactured by Myoridge, lot numbers A20, C04, C23)
・ iMatrix-511 silk (product number 387-10131, manufactured by Nippi, lot number S19F008)
・CultureSure ^™ Y-27632 (product number 034-24024, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., lot number KCG7025)
・ Cell detachment solution (0.25% Trypsin EDTA, product number 25200-072, manufactured by gibco)

<Culture schedule>
Cell culture was performed according to the following schedule. The day after cell wakeup is defined as Day 0, and subsequent days are represented.
Day-1: Reagent preparation and preparation Day0: Frozen cardiomyocytes Day1-4: Pre-culture Day5: Seeding in 24-well plate for test Day12: Cardiomyocyte sampling

<iMatrix-511 silk coating method for 24-well plate for test>
74 μL of iMatrix-511 silk was added to 9.5 mL of PBS(−) for dilution. This was added to a test 24-well plate at 500 μL/well (concentration used: 1.03 μg/cm ² ) and allowed to stand in a 37° C. incubator for 1 hour.

<Sleeping of frozen cardiomyocytes>
Y-27632 was added to a seeding medium for CarmyA to a final concentration of 10 μM and used as a thawing medium. It was used after warming to 37 degreeC at the time of use.
Frozen myocardial cells were thawed in a 37° C. water bath and suspended in a thawing medium. Centrifuge at 300×g for 5 minutes and remove the supernatant. Cells were suspended in a thawing medium, adjusted to 2×10 ⁶ to 5×10 ⁶ cells/ml, then counted, and 3×10 ⁵ to 8×10 ⁵ cells/cm ² in a T25 flask (Product No. 353014, manufactured by FALCON, culture area 25 cm ² ). Flasks were placed in a 37° C., 5% CO ₂ incubator. The day after seeding, the entire medium was replaced with CarmyA maintenance medium at 37°C.

<Pre-culture>
The medium was exchanged every day, and the whole amount was exchanged with CarmyA maintenance medium at 37°C.

<Seeding cardiomyocytes into 24-well plate for testing>
After the precultured cells were washed with PBS(-), a cell detachment solution was added and incubated at 37°C for 5 to 10 minutes to detach the cells. The detached cells were suspended in a seeding medium for CarmyA to which Y-27632 was added to a final concentration of 3 μM, and the number of cells was pre-counted. Centrifuge at 300×g for 5 minutes and remove the supernatant. The cells were suspended in a seeding medium for CarmyA to which Y-27632 was added to a final concentration of 10 μM, adjusted to 2×10 ⁵ to 5×10 ⁵ cells/ml based on the pre-counted cell number, and then resuspended. Cell number was counted. Using this number of cells, cells were seeded in a 24-well plate for testing at 1.63×10 ⁵ cells/well and allowed to stand in a 37° C., 5% CO ₂ incubator. On the next day after seeding, the medium was exchanged, and the whole amount was exchanged with a maintenance medium for CarmyA at 37°C. Thereafter, the entire medium was replaced with CarmyA maintenance medium at 37°C every 1 to 2 days. Experiments were performed in triplicate.

<RNA extraction and RT-qPCR>
cTnT, MYL2, ATP2A2, PPARGC1A, PPARA, SLC2A4, BNP and GAPDH were analyzed.

(1) Reagents and instruments RNA extraction: miRNeasy Mini Kit (product number 217004, manufactured by Qiagen)
cDNA synthesis: ReverTra Ace (R) qPCR RT Master Mix with gDNA Remover (product number FSQ-301, manufactured by TOYOBO)
qPCR reaction: PowerUp SYBR Green Master Mix (product number A25776, Thermo Fisher)
QuantStudio 6 Flex Real-time PCR system (manufactured by Thermo Fisher)
Nanophotometer spectrophotometer C40 (manufactured by Wakenby Tech Co., Ltd.)

(2) RNA Extraction After removing the culture supernatant of cardiomyocytes, 0.5 mL of QIAZOL (manufactured by Qiagen) was added and suspended to lyse the cells, and the lysate was collected in a 1.5 ml tube. After that, RNA was extracted according to the protocol attached to the miRNeasy Mini Kit, and the RNA concentration was measured with a Nanophotometer C40.

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

<Evaluation of gene expression>
Using the RNA extracted from the cells, gene expression levels were analyzed using a QuantStudio 6 Flex Real-time PCR system. Table 7 shows the sequences of the primers used, and Table 8 shows the PCR conditions. The gene expression level is shown as a relative value when the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is set to 1. The results are shown in FIGS. 1-7.

From FIG. 1, the expression level of cTnT on the T plate (Examples 1 and 2) was higher than that on Corning (Comparative Examples 1 and 2).
From FIG. 2, the expression level of Myl2 on the T plate (Examples 1 and 2) was higher than that on Corning (Comparative Examples 1 and 2).
From FIG. 3, the expression level of ATP2A2 on the T plate (Examples 1 and 2) was higher than that on Corning (Comparative Examples 1 and 2).
From FIG. 4, the expression level of PPARGC1A on the T plate (Examples 1 and 2) was higher than that on Corning (Comparative Examples 1 and 2).
From FIG. 5, the expression level of PPARA on the T plate (Examples 1 and 2) was higher than that on Corning (Comparative Examples 1 and 2).
From FIG. 6, the expression level of SLC2A4 on the T plate (Examples 1 and 2) was higher than that on Corning (Comparative Examples 1 and 2).
From FIG. 7, the expression level of BNP on the T plate (Examples 1 and 2) was lower than that on Corning (Comparative Examples 1 and 2).

From the above results, the cardiomyocyte function can be improved by using the cardiomyocyte culture method of the present invention.

Claims (13)

A method for culturing cardiomyocytes, comprising a step (A) of seeding cardiomyocytes on a culture surface of a culture vessel and a step (B) of culturing the cardiomyocytes in a medium, wherein at least the culture surface of the culture vessel A method for culturing cardiomyocytes, a part of which is formed from a substrate containing a 4-methyl-1-pentene polymer. The 4-methyl-1-pentene polymer is at least one copolymer selected from 4-methyl-1-pentene, ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). The method for culturing cardiomyocytes according to claim 1, wherein the cardiomyocytes are polymers. 3. The method for culturing cardiomyocytes according to claim 1, wherein the entire culture surface of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer. The method for culturing cardiomyocytes according to any one of claims 1 to 3, wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells. 5. The method for culturing cardiomyocytes according to claim 4, wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells by a protein-free cardiomyocyte differentiation induction (PFCD) method. 6. The method for culturing cardiomyocytes according to claim 4 or 5, wherein the expression level of at least one gene selected from a cardiomyocyte marker and a sarcoplasmic reticulum marker is increased. The myocardium according to claim 6, wherein the cardiomyocyte marker is at least one selected from cardiac troponin T (cardiac muscle) and MLC-2 (Myosin regulatory light chain 2, ventricular/cardiac muscle isoform). Cell culture method. The method for culturing cardiomyocytes according to claim 6, wherein the sarcoplasmic reticulum marker is SERCA2 (sarcoplasmic/endoplasmic reticulum calcium ATPase 2). 6. The method for culturing cardiomyocytes according to any one of claims 1 to 5, wherein expression levels of genes of factors involved in energy metabolism control are increased. 前記エネルギー代謝制御に関与する因子が、ＰＧＣ－１α（Ｐｅｒｏｘｉｓｏｍｅ ｐｒｏｌｉｆｅｒａｔｏｒ－ａｃｔｉｖａｔｅｄ ｒｅｃｅｐｔｏｒ ｇａｍｍａ ｃｏａｃｔｉｖａｔｏｒ １－ａｌｐｈａ）、ＰＰＡＲα（Ｐｅｒｏｘｉｓｏｍｅ ｐｒｏｌｉｆｅｒａｔｏｒ－ａｃｔｉｖａｔｅｄ ｒｅｃｅｐｔｏｒ ａｌｐｈａ）、及びＧＬＵＴ‐４（Ｓｏｌｕｔｅ ｃａｒｒｉｅｒ ｆａｍｉｌｙ ２，ｆａｃｉｌｉｔａｔｅｄ ｇｌｕｃｏｓｅ ｔｒａｎｓｐｏｒｔｅｒ ｍｅｍｂｅｒ 10. The method for culturing cardiomyocytes according to claim 9, wherein the cardiomyocyte is at least one selected from 4). 6. The method for culturing cardiomyocytes according to any one of claims 1 to 5, wherein the expression level of a cardiomyocyte ischemia marker gene is decreased. The method for culturing cardiomyocytes according to claim 11, wherein the cardiomyocyte ischemia marker is BNP (brain natural peptide). The method for culturing cardiomyocytes according to any one of claims 1 to 12, wherein the maximum vertical distance from the culture surface of the culture vessel to the upper surface of the medium is greater than 7 mm.

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