WO2015199118A1 - Method for promoting phosphorylation of erk or akt in cultured cell, cell culture method, and phosphorylation promoter - Google Patents

Abstract

Provided are: a method for promoting phosphorylation of an extracellular signal-regulated kinase (ERK) or an AKT in a cultured cell; a cell culture method using the method for promoting phosphorylation; and a phosphorylation promoter appropriately usable in these methods. Phosphorylation of an ERK or an AKT in a cultured cell can be promoted by contacting the cultured cell with a molding of an alicyclic structure-containing polymer.

Description

Method for enhancing phosphorylation of ERK or AKT in cultured cells, cell culture method, and phosphorylation enhancer

The present invention relates to a method for enhancing phosphorylation of ERK (Extracellular signal-Regulated Kinase) or AKT in cultured cells, a method for culturing cells using this method for enhancing phosphorylation, and a phosphorylation enhancer suitably used for these methods. About.

When culturing adherent cells using a culture container such as a dish or flask, if the cells proliferate and the bottom surface of the culture container is completely covered with cells, the newly generated cells by cell division Because it cannot adhere to the bottom of the skin, it causes apoptosis. As a result, the number of living cells decreases and the contents leak from the cells that have undergone apoptosis.
The contents leaked from the apoptotic cells (leakage contaminants) may affect the assay using the cells. In addition, when a substance such as a medicine is produced using cells, there is a problem that leakage contaminants cause decomposition of the product or increase the number of work steps for the removal.
For this reason, it has been required to efficiently suppress apoptosis in cell culture.

Apoptosis is known to be caused by the action of multiple types of proteolytic enzymes in cells called caspases. In addition, it is known that what causes the caspase to activate is leakage of cytochrome or the like from mitochondria.
Non-Patent Document 1 describes that leakage of contents from mitochondria is caused by an information transmission action via a protein related to cell surface adhesion called integrin if the adherent cell is adhered to an extracellular matrix. It is described that it has an action to prevent, and as a result, apoptosis is prevented.
Regarding the action mechanism of apoptosis suppression in adherent cells, phosphorylation of intracellular signal transduction system protein called ERK or AKT in cells via integrins is enhanced, and the phosphorylation of ERK or AKT is enhanced. It is known that the action brings about an effect of making a factor that destroys mitochondria non-destructive.

Patent Document 1 discloses that a human-derived CAP18 partial peptide or an antimicrobial peptide such as defensin prevents cell death (apoptosis). Specifically, when neutrophil cells, which are floating cells, are cultured in a cell culture medium supplemented with a partial peptide of human-derived CAP18 or defensin, decreased caspase activity and increased phosphorylation of ERK are observed. This partial peptide is described as having an inhibitory effect on apoptosis. In this document, a partial peptide of CAP18 has a binding action on the FPRL-1 receptor expressed on the cell surface of neutrophil cells, and defensin is mediated through the CCR6 receptor, which is a chemokine receptor. It is also described that it can be obtained by acting. However, given this mechanism of action, the anti-apoptotic effect requires that a receptor capable of specific binding corresponding to the component be expressed on the cell surface, and of course, the effect is limited by the type of cell. Is done.
Furthermore, Patent Document 2 discloses that a compound having a pyridazinone structure has caspase inhibitory activity in vivo against floating cells such as Jurkat cells derived from T cells in blood cells. .

By the way, as a cell culture instrument, since it is light and transparent, the product made from polystyrene is used widely as a commercial product. In addition, as a culture container using a material other than polystyrene, a fluorine-substituted product such as polyethylene (Patent Document 3) and a culture container obtained using a cycloolefin resin (Patent Document 4) are foreign substances to the medium, Since there is no elution, it is known that the culture performance is excellent.
These culture devices are usually sterilized by a treatment such as γ-ray irradiation. Further, the surface of the culture vessel may be subjected to a hydrophilic treatment by plasma treatment or ultraviolet irradiation treatment so that the adherent cells do not peel off and die.

JP 2007-169260 A JP2009-545585 (US2009 / 0291959) Japanese Patent Laying-Open No. 2005-218444 (US2005 / 0153438) JP 2009-027944 A

As described above, although various techniques for preventing apoptosis of cultured cells have been proposed so far, there is a demand for a more efficient and more versatile method for preventing apoptosis. It is.
The present invention has been made in view of the state of the prior art, and is suitable for a method for enhancing phosphorylation of ERK or AKT in cultured cells, a method for culturing cells using this method for enhancing phosphorylation, and these methods. An object of the present invention is to provide a phosphorylation-enhancing agent to be used in

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that phosphorylation of ERK and AKT can be enhanced by bringing a cultured cell into contact with an alicyclic structure-containing polymer molded product. It came to be completed.

Thus, according to the present invention, there are provided the following phosphorylation enhancing methods (1) to (3), (4) cell culturing methods, and (5) and (6) phosphorylation enhancing agents.
(1) A method for enhancing phosphorylation of ERK (Extracellular signal-Regulated Kinase) in cultured cells, which comprises contacting a cultured cell with an alicyclic structure-containing polymer molded product.
(2) A method for enhancing phosphorylation of AKT in cultured cells, which comprises contacting a cultured cell with an alicyclic structure-containing polymer molded product.
(3) The method for enhancing phosphorylation according to (1) or (2), wherein the cultured cells are adherent cells.
(4) Contacting a cultured cell with an alicyclic structure-containing polymer molded article to enhance phosphorylation of ERK (Extracellular signal-Regulated Kinase) and / or phosphorylation of AKT in the cultured cell A cell culture method.
(5) An agent for enhancing phosphorylation of intracellular ERK in cultured cells, comprising an alicyclic structure-containing polymer molded product.
(6) An agent for enhancing phosphorylation of intracellular AKT in cultured cells, comprising an alicyclic structure-containing polymer molded product.

According to the present invention, there are provided a method for enhancing phosphorylation of ERK or AKT in cultured cells, a method for culturing cells using this method for enhancing phosphorylation, and a phosphorylation enhancer suitably used for these methods.

FIG. 1 is a graph of the relative value of phosphorylation of ERK-1 in CHO cells. FIG. 2 is a graph of the relative value of phosphorylation of ERK-2 in CHO cells. FIG. 3 is a graph of the relative value of phosphorylation of ERK-1 in VERO cells. FIG. 4 is a graph of the relative value of phosphorylation of ERK-2 in VERO cells. FIG. 5 is a graph of the relative value of AKT phosphorylation in CHO cells. FIG. 6 is a graph showing the number of viable cells when CHO cells are cultured for 3 weeks. FIG. 7 is a graph showing the number of viable cells when VERO cells are cultured for 3 weeks. FIG. 8 is a graph of relative values of LDH values in the culture medium when CHO cells are cultured for 3 weeks. FIG. 9 is a graph of the relative values of LDH values in the culture medium when VERO cells were used for 3 weeks. FIG. 10 is a graph of the relative value of phosphorylation of p44 ERK in CHO cells when the sterilization method of the culture container is changed. FIG. 11 is a graph of the relative value of phosphorylation of p42 ERK of CHO cells when the sterilization method of the culture container is changed.

The cells used in the present invention are not particularly limited, and can be arbitrarily selected according to the purpose. Of these, adherent cells are preferred because the effects of the present invention are more easily obtained. In the present invention, the adherent cell may be an adherent cell itself or a cell derived from an adherent cell. Adhesive cells themselves are cells that can survive and proliferate by adhering to an extracellular matrix under normal culture conditions, and are also referred to as anchorage-dependent cells. Adherent cell-derived cells adhere to the extracellular matrix by applying some external factor to the adherent cells, such as cells that have been acclimated and cultured in the cultivated adherent cells, and that have survived and can proliferate. It is a cell that can survive and proliferate without it. Examples of the adherent cells include genetically engineered host cells and virus-sensitive cells such as CHO cells, VERO cells, NIH3T3 cells, HEK293 cells and the like.

When culturing cells, a liquid medium is usually used.
As the liquid medium, a medium having a pH buffering action, having an osmotic pressure suitable for cells, containing nutrient components of cells, and not toxic to cells is used.
Examples of the component exhibiting pH buffering action include tris hydrochloride, various phosphates, and various carbonates.
The osmotic pressure of the liquid medium is usually adjusted using an aqueous solution in which the concentrations of potassium ions, sodium ions, calcium ions, glucose and the like are adjusted so as to be almost the same as the osmotic pressure of the cells. Specific examples of the aqueous solution include physiological saline such as phosphate buffered saline, Tris buffered saline, and HEPES buffered saline; Ringer's solution such as lactated Ringer's solution, acetated Ringer's solution, and bicarbonated Ringer's solution; It is done.
Examples of the nutrient component of the cell include amino acids, nucleic acids, vitamins, minerals and the like.
As the liquid medium, various commercial products such as RPMI-1640, HAM, α-MEM, DMEM, EMEM, F-12, F-10, and M-199 can be used.

An additive can also be mix | blended with a liquid culture medium. Examples of additives include differentiation-inducing factors such as proteins, low-molecular compounds having differentiation-inducing activity, minerals, metals, and vitamin components.
Differentiation inducing factors include ligands, agonists and antagonists that act on cell surface receptors; nuclear receptor ligands, agonists and antagonists; extracellular matrices such as collagen and fivenectin; parts of the extracellular matrix; Compounds that mimic extracellular matrix; Components that act on proteins involved in intracellular signal transduction pathways; Components that act on enzymes of primary or secondary metabolism in cells; Genes of genes in intracellular nucleus or mitochondria Ingredients that affect expression; DNA such as DNA encoding a cell growth factor gene such as insulin-like growth factor, or RNA such as microRNA designed to have an interfering RNA effect on intracellular regulators such as caspatase, Injection method, hydrodynamic Law, electroporation method, DNA or RNA can be introduced in combination with such a viral vector into cells by a method such as lipofectin method, and the like.
These additives can be used alone or in combination of two or more.

The cell culture conditions are not particularly limited, and can be appropriately determined according to the cells to be used and the purpose.
For example, the cells can be cultured using a humidified thermostat having a carbon dioxide concentration of about 5% and a constant temperature in the range of 20 ° C. to 37 ° C.

The alicyclic structure-containing polymer molded product used in the present invention is formed by molding an alicyclic structure-containing polymer into an arbitrary shape.
The alicyclic structure-containing polymer is a resin having an alicyclic structure in the main chain and / or side chain, and preferably contains an alicyclic structure in the main chain from the viewpoint of mechanical strength, heat resistance, and the like.

Examples of the alicyclic structure include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. From the viewpoint of mechanical strength, heat resistance, etc., a cycloalkane structure or a cycloalkene structure. A structure is preferable, and a structure having a cycloalkane structure is most preferable.

The number of carbon atoms constituting the alicyclic structure is not particularly limited, but is usually 4 to 30, preferably 5 to 20, and more preferably 5 to 15. When the number of carbon atoms constituting the alicyclic structure is within this range, mechanical strength, heat resistance, and moldability are highly balanced, which is preferable.

The proportion of the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer may be appropriately selected according to the purpose of use, but is usually 30% by weight or more, preferably 50% by weight or more, more preferably 70% by weight. %. If the proportion of the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer is excessively small, the heat resistance is inferior, which is not preferable. The remainder other than the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer is not particularly limited and is appropriately selected according to the purpose of use.

Specific examples of the alicyclic structure-containing polymer include (1) norbornene polymer, (2) monocyclic olefin polymer, (3) cyclic conjugated diene polymer, and (4) vinyl alicyclic carbonization. Examples thereof include hydrogen polymers and hydrides of (1) to (4). Among these, norbornene-based polymers and hydrides thereof are preferable from the viewpoints of heat resistance, mechanical strength, and the like.

(1) Norbornene-based polymer The norbornene-based polymer is obtained by polymerizing a norbornene-based monomer that is a monomer having a norbornene skeleton, and is obtained by ring-opening polymerization or by addition polymerization. Broadly divided into things.

Examples of the ring-opening polymer obtained by ring-opening polymerization include ring-opening polymers of norbornene monomers, ring-opening polymers of norbornene monomers and other monomers capable of ring-opening copolymerization, and these A hydride etc. are mentioned. Examples of those obtained by addition polymerization include addition polymers of norbornene monomers and addition polymers of norbornene monomers and other monomers copolymerizable therewith. Among these, a ring-opening polymer hydride of a norbornene-based monomer is preferable from the viewpoint of heat resistance, mechanical strength, and the like.

Examples of the norbornene monomer include bicyclo [2.2.1] hept-2-ene (common name: norbornene), 5-methyl-bicyclo [2.2.1] hept-2-ene, 5,5-dimethyl. -Bicyclo [2.2.1] hept-2-ene, 5-ethyl-bicyclo [2.2.1] hept-2-ene, 5-ethylidene-bicyclo [2.2.1] hept-2-ene 5-vinyl-bicyclo [2.2.1] hept-2-ene, 5-propenylbicyclo [2.2.1] hept-2-ene, 5-methoxycarbonyl-bicyclo [2.2.1] hepta Bicyclic single units such as -2-ene, 5-cyanobicyclo [2.2.1] hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo [2.2.1] hept-2-ene Mer;
Tricyclo [4.3.0 ^1,6 . 1 ^2,5 ] deca-3,7-diene (common name dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, etc. Mer;
Tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene (tetracyclododecene), tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidenetetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8,9-dimethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyl-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidene-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyl-8-carboxymethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene,
7,8-benzotricyclo [4.3.0.1 ^2,5 ] dec-3-ene (common name methanotetrahydrofluorene: also called 1,4-methano-1,4,4a, 9a-tetrahydrofluorene) 1,4-methano-8-methyl-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8 -Tetracyclic monomers such as bromo-1,4,4a, 9a-tetrahydrofluorene;

Other monomers capable of ring-opening copolymerization with norbornene monomers include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, And monocyclic cycloolefin monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatetraene.
These monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

Other monomers capable of addition copolymerization with norbornene monomers include α-olefin monomers having 2 to 20 carbon atoms such as ethylene, propylene, 1-butene, 1-pentene and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo ^{[9.2.1.0 2,10.} Cycloolefin monomers such as 0 ^3,8 ] tetradeca-3,5,7,12-tetraene (also referred to as 3a, 5,6,7a-tetrahydro-4,7-methano-1H-indene); Non-conjugated diene monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, 1,7-octadiene, and the like.
Among these, as other monomers capable of addition copolymerization with norbornene monomers, α-olefin monomers are preferable, and ethylene is more preferable.
These monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization with a monomer component is a known ring-opening polymerization. It can be obtained by polymerization in the presence of a catalyst. Examples of the ring-opening polymerization catalyst include a catalyst comprising a metal halide such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide or acetylacetone such as titanium, zirconium, tungsten, or molybdenum. A catalyst comprising a compound and an organoaluminum compound can be used.
The ring-opening polymer hydride of a norbornene-based monomer is usually obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to the polymerization solution of the ring-opening polymer and then adding a carbon-carbon unsaturated bond. Can be obtained by hydrogenation.

An addition polymer of a norbornene monomer, or an addition polymer of a norbornene monomer and another monomer copolymerizable with the norbornene monomer, in the presence of a known addition polymerization catalyst. It can be obtained by polymerization. As the addition polymerization catalyst, for example, a catalyst composed of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

(2) Monocyclic cyclic olefin polymer As the monocyclic olefin polymer, for example, an addition polymer of a monocyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used. it can.
(3) Cyclic conjugated diene polymer As the cyclic conjugated diene polymer, for example, a polymer obtained by subjecting a cyclic conjugated diene monomer such as cyclopentadiene or cyclohexadiene to 1,2- or 1,4-addition polymerization, and The hydride can be used.
(4) Vinyl alicyclic hydrocarbon polymer Examples of the vinyl alicyclic hydrocarbon polymer include polymers of vinyl alicyclic hydrocarbon monomers such as vinyl cyclohexene and vinyl cyclohexane and their hydrides; And hydrides of aromatic ring portions of polymers of vinyl aromatic monomers such as α-methylstyrene. The vinyl alicyclic hydrocarbon polymer may be a copolymer with other monomers copolymerizable with these monomers.

Although there is no special restriction | limiting in the molecular weight of an alicyclic structure containing polymer, it is a weight average molecular weight of polystyrene conversion measured by the gel permeation chromatography of a cyclohexane solution (a toluene solution when a polymer does not melt | dissolve), and is usually 5 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and particularly preferably 10,000 to 100,000. When the weight average molecular weight is within this range, the mechanical strength and the moldability are highly balanced, which is preferable.

The glass transition temperature of the alicyclic structure-containing polymer may be appropriately selected depending on the purpose of use, but is usually 50 to 300 ° C, preferably 100 to 280 ° C, particularly preferably 115 to 250 ° C, and more preferably 130. ~ 200 ° C. When the glass transition temperature is within this range, heat resistance and molding processability are highly balanced and suitable.
In the present invention, the glass transition temperature is measured based on JIS K7121.

These alicyclic structure-containing polymers can be used alone or in combination of two or more.
In addition, for the alicyclic structure-containing polymer, a compounding agent usually used in thermoplastic resin materials, for example, a soft polymer, an antioxidant, an ultraviolet absorber, a light stabilizer, a near infrared absorber, a release agent. Additives such as colorants such as dyes and pigments, plasticizers, antistatic agents, fluorescent brighteners, and the like can be added in amounts that are usually employed.
In addition, the alicyclic structure-containing polymer may be mixed with another polymer other than the soft polymer (hereinafter simply referred to as “other polymer”). The amount of the other polymer mixed with the alicyclic structure-containing polymer is usually 200 parts by weight or less, preferably 150 parts by weight or less, more preferably 100 parts by weight with respect to 100 parts by weight of the alicyclic structure-containing polymer. It is as follows.
When the proportion of various compounding agents and other polymers to be blended with respect to the alicyclic structure-containing polymer is too high, the ability to enhance phosphorylation of intracellular signal transduction proteins decreases. It is preferable to mix in the range which does not impair the property.
The mixing method with the compounding agent and other polymers is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Moreover, there is no special restriction | limiting in the order of a mixing | blending. As a blending method, for example, a method of kneading a resin in a molten state using a mixer, a uniaxial kneader, a biaxial kneader, a roll, a Brabender, an extruder, etc., after dissolving and dispersing in a suitable solvent, Examples thereof include a method of removing the solvent by a coagulation method, a casting method, or a direct drying method.
When a biaxial kneader is used, after kneading, it is usually extruded in a rod shape in a molten state, cut into an appropriate length with a strand cutter, and pelletized in many cases.

The shaping | molding method of an alicyclic structure containing polymer can be arbitrarily selected according to the shape of the alicyclic structure containing polymer molded object used when making it contact with a cell. Examples of molding methods include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendar molding, and rolling molding. , Cutting molding method, spinning and the like, and these molding methods can be combined, or post-treatment such as stretching can be carried out as necessary after molding.
The molded product thus obtained is the ERK or AKT phosphorylation enhancer of the present invention.

There is no special restriction | limiting in the shape of an alicyclic structure containing polymer molded object, A plate shape, a powder form, a granular form, a string form, a sheet form, and other shapes may be sufficient. Moreover, the surface may be flat, may have an uneven shape, or may be a hollow molded body. Different shaped bodies can be combined into another shaped body with or without an adhesive or the like.
In addition, culture containers such as dishes, plates, bags, tubes, scaffolds, cups, jars and fermenters; parts of culture devices such as stirring blades, stirrers, baffles, and connecting tubes; It may be a member constituting part or all of a culture instrument used for culture operation such as a pipette, a stirring element, a filter, and a cell scraper.

In the present invention, it is preferable to sterilize the molded body when the molded body is brought into contact with the cultured cells. There are no particular restrictions on the method of sterilization, heating methods such as the high-pressure steam method and dry heat method; radiation methods that irradiate radiation such as γ rays and electron beams; irradiation methods that irradiate high frequencies; ethylene oxide gas (EOG) From a method generally employed in the medical field such as a gas method in which a gas is brought into contact with each other, it can be selected according to the shape of the molded body and the cells to be used. A gas method in which a gas such as ethylene oxide gas is brought into contact is preferred because of its high phosphorylation enhancing activity.
In addition, the surfaces of these molded bodies can be subjected to treatments other than the sterilization purpose generally applied to culture vessels such as plasma treatment, corona discharge treatment, ozone treatment, ultraviolet irradiation treatment, etc., but phosphorylation of ERK and AKT From the viewpoint of the acceleration rate, it is preferable to use these treatments without performing them.

As the method for bringing the cultured cell into contact with the polymer molded product containing an alicyclic structure which is an ERK or AKT phosphorylation enhancer of the present invention, any method may be adopted depending on the shape of the ERK or AKT phosphorylation enhancer. That's fine. For example, a method of culturing cells in a medium in which an alicyclic structure-containing polymer molded body that is an ERK or AKT phosphorylation enhancer is mixed; a cell is cultured in a culture vessel formed using the alicyclic structure-containing polymer. Examples include a method of culturing; a method of performing a culturing operation using a culture device formed using an alicyclic structure-containing polymer, and the like.
In addition, since the cells have the ability to transmit information, it is not necessary for all cultured cells in culture to be in contact with the polymer molded body containing an alicyclic structure, and both are in contact throughout the entire culture period. There is no need. However, since the effect of contact decreases with time, a longer contact time is preferable.
The contact temperature between the cultured cell and the alicyclic structure-containing polymer molded product is not particularly limited as long as the cell can grow.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.
[Production Example 1]
As an alicyclic structure-containing polymer, ZEONEX (registered trademark) 790R (manufactured by Nippon Zeon Co., Ltd., norbornene-based ring-opening polymer hydride; hereinafter simply referred to as “790R”) was used, and a petri dish having a diameter of 35 mm was formed by injection molding. A shaped culture vessel was obtained and then sterilized with ethylene oxide. Hereinafter, this culture container is referred to as “790R container”.

[Production Example 2]
In Production Example 1, instead of 790R, ZEONOR (registered trademark) 1430R (manufactured by Nippon Zeon Co., Ltd., norbornene-based ring-opening polymer hydride; hereinafter simply referred to as “1430R”) was used. A culture vessel was obtained in the same manner. Hereinafter, this culture container is referred to as a “1430R made container”.

[Production Example 3]
In Production Example 1, instead of 790R, Zeonore (registered trademark) 1060R (manufactured by Nippon Zeon Co., Ltd., norbornene-based ring-opening polymer hydride; hereinafter simply referred to as “1060R”) was used. A culture vessel was obtained in the same manner. Hereinafter, this culture container is referred to as “1060R-made container”.

[Production Example 4]
In order to facilitate adhesion of adherent cells, plasma irradiation was performed as described below to produce a culture vessel having a surface hydrophilized.
First, using a 790R, a petri dish-shaped culture vessel having a diameter of 35 mm was obtained by injection molding, and then plasma irradiation was performed to hydrophilize the vessel surface. Subsequently, this was subjected to sterilization with ethylene oxide. Hereinafter, this culture container is referred to as “surface hydrophilized 790R container”.

[Production Example 5]
In Production Example 4, a culture vessel was obtained in the same manner as in Production Example 4 except that 1430R was used instead of 790R. Hereinafter, this culture container is referred to as “surface hydrophilized 1430R container”.

[Example 1]
CHO cells were seeded at a cell density of 1.25 × 10 ⁴ cells / cm ² in a 790R container containing 2 ml of medium, placed in a CO ₂ incubator set at a temperature of 37 ° C. and a CO ₂ concentration of 5%, and cultured for 5 days. Then, ERK phosphorylation was analyzed by the method described later.

[Example 2]
In Example 1, in place of the 790R container, the culture was performed in the same manner as in Example 1 except that the 1430R container was used, and ERK phosphorylation was analyzed.

Example 3
In Example 1, in place of the 790R container, culturing was performed in the same manner as in Example 1 except that a surface hydrophilized 790R container was used, and ERK phosphorylation was analyzed.

Example 4
In Example 1, in place of the 790R container, the culture was performed in the same manner as in Example 1 except that a surface hydrophilized 1430R container was used, and ERK phosphorylation was analyzed.

[Comparative Example 1]
In Example 1, instead of a 790R container, a commercially available polystyrene dish [Falcon (registered trademark) dish (Becton Dickinson, model number 353001)] (hereinafter referred to as “polystyrene container”) Except that was used, the culture was carried out in the same manner as in Example 1, and the phosphorylation of ERK was analyzed.

(Analysis of ERK phosphorylation)
To the cell sample collected from the 790R container, an electrophoresis buffer containing SDS (sodium dodecyl sulfate) having a protein denaturing action was added and heated at 100 ° C. for 5 minutes to dissolve the cell sample. . This was allowed to stand at 4 ° C. for 5 minutes, followed by centrifugation to remove insolubles by precipitation to prepare a sample for electrophoresis.
Similarly, electrophoresis samples were prepared using cells cultured in a 1430R container, a surface hydrophilized 790R container, a surface hydrophilized 1430R container, and a polystyrene container.
Two samples for electrophoresis were prepared.
Each obtained electrophoresis sample was applied to an electrophoresis sample comb of a precast gel (manufactured by Nacalai Tesque), applied with voltage, and subjected to SDS-polyacrylamide electrophoresis.

Next, the acrylamide gel subjected to the electrophoresis operation was taken out, and the protein that had been electrophoresed in the acrylamide gel was transferred to a nitrocellulose membrane (CST) using a Western blotting transfer device (Nihon Aido).
Blocking of skim milk in Tris phosphate buffer solution (hereinafter referred to as “buffer solution TBS-T”) containing Tween (registered trademark) 20 having a final concentration of 0.05% on the nitrocellulose membrane after transfer. The nitrocellulose membrane surface was blocked by immersing in the solution and shaking at room temperature for 1 hour. Subsequently, the nitrocellulose membrane was washed by immersing in buffer solution TBS-T for 5 minutes, and this washing operation was repeated three times.
Prepare a solution in which anti-ERK antibody (manufactured by CST), which is an antibody specifically detecting ERK1 and ERK2, is added as a primary antibody to buffer TBS-T containing 5% BSA as a primary antibody. Then, the nitrocellulose membrane was immersed in this solution for 16 hours.
Similarly, a solution in which anti-phosphorylated ERK antibody (manufactured by CST), which is an antibody specifically detecting phosphorylated ERK1 and phosphorylated ERK2, is added as a primary antibody to buffer TBS-T containing 5% BSA. A nitrocellulose membrane was immersed in this solution for 16 hours.

The nitrocellulose membrane immersed in the anti-ERK antibody solution and the nitrocellulose membrane immersed in the anti-phosphorylated ERK antibody solution were each immersed in the buffer solution TBS-T for 5 minutes and washed, and this washing operation was repeated three times. Subsequently, a nitrocellulose membrane immersed in an anti-ERK antibody solution and washed in a buffer solution TBS-T containing 5% BSA containing a secondary antibody for detecting the primary antibody on the surface of the nitrocellulose membrane, and anti-phosphorus The nitrocellulose membrane immersed and washed in the oxidized ERK antibody solution was immersed and shaken at room temperature for 1 hour.
After the shaking treatment, washing was performed by immersing in buffer solution TBS-T for 5 minutes, and this washing operation was repeated three times.
The membrane was immersed in a detection color reagent 1-Step-Ultra TMB Blotting Solution (Pierce), and a color reaction was performed on the membrane, thereby detecting an immune reaction signal of ERK or phosphorylated ERK.

The nitrocellulose membrane color-treated with the immune reaction signal was photographed with a digital camera (manufactured by Ricoh), and the signal intensity of the immune reaction was digitized using ImageJ for the obtained image.
In order to compare the phosphorylation effect of ERK, the value obtained by dividing the reaction signal value of anti-phosphorylated ERK antibody by the reaction signal value of anti-ERK antibody (total ERK) was obtained for each container. The respective values in the 790R container, the surface hydrophilized 790R container, the 1430R container, and the surface hydrophilized 1430R container are obtained when the numerical value in the comparison polystyrene container is the standard (ie, 1). It was.

As shown in FIG. 1, phosphorylation activation of ERK-1 in CHO cells cultured in 790R and 1430R containers is more than 4 times that of CHO cells cultured in polystyrene containers, and the cells are alicyclic. It has been shown that phosphorylation of ERK-1 is enhanced by contact with a structure-containing polymer.
In the culture using the surface hydrophilized 790R container hydrophilized 790R surface and the 1430R hydrophilized surface hydrophilized 1430R container, the phosphorylation is twice as high as the culture using the polystyrene container. It was observed.

Regarding phosphorylation of ERK-2, as shown in FIG. 2, when a 790R container was used, phosphorylation was confirmed to be about 4 times higher than when a polystyrene container was used. When used, an increase in phosphorylation of about 5 times was confirmed.
Further, when the surface hydrophilized 790R container and the surface hydrophilized 1430R container were used, ERK-2 phosphorylation was doubled as compared with the case of using the polystyrene container.

Example 5
In Example 1, except that VERO cells were used instead of CHO cells, culture was performed using a 790R container in the same manner as in Example 1, and ERK phosphorylation was analyzed.

Example 6
In Example 5, except that a 1430R container was used instead of the 790R container, culture was performed in the same manner as in Example 5, and ERK phosphorylation was analyzed.

[Comparative Example 2]
In Example 5, except that a polystyrene container was used instead of the 790R container, culture was performed in the same manner as in Example 5, and ERK phosphorylation was analyzed.

As shown in FIG. 3, in the VERO cells cultured in the 790R container, phosphorylation of ERK-1 was increased about twice as much as when the polystyrene container was used, and the 1430R container was used. It was confirmed that phosphorylation of ERK-1 was increased about twice or more.
As shown in FIG. 4, regarding the phosphorylation of ERK-2, when a 790R container or a 1430R container is used, both are about 1.5 times as much as when a polystyrene container is used. Increased.
Thus, like CHO cells, VERO cells were shown to have an effect of enhancing phosphorylation of ERK by contacting with an alicyclic structure-containing polymer.

Example 7
Cells were cultured under the same conditions as in Example 1, and then AKT phosphorylation was analyzed by the method described below.

Example 8
In Example 7, except that a 1060R container was used in place of the 790R container, culture was performed in the same manner as in Example 7, and AKT phosphorylation was analyzed.

[Comparative Example 3]
In Example 7, it culture | cultivated like Example 7 except having replaced with the container made from 790R, and using the container made from polystyrene, and analyzed phosphorylation of AKT.

(AKT phosphorylation analysis)
In the method for analyzing phosphorylation of ERK shown above, an anti-AKT antibody was used in place of the anti-ERK antibody, and an anti-phosphorylated AKT antibody was used in place of the anti-phosphorylated ERK antibody. Intracellular AKT phosphorylation was analyzed by a method similar to the phosphorylation analysis method.
As shown in FIG. 5, regarding the phosphorylation of AKT in CHO cells, when a 790R container or a 1060R container is used, both are 1.5 times as much as when a polystyrene container is used. A degree of enhancement was seen.

[Production Example 6]
A 1430R film was heat-bonded to the bottom of a Pyrex (registered trademark) glass tube having an inner diameter of 20 mm, a thickness of 1 mm, and a length of 18 mm, and this was subjected to γ-ray sterilization to obtain a culture cup. This cup is referred to as a “1430R cup”.
[Production Example 7]
In Production Example 6, a cup having a hydrophilic surface was obtained in the same manner as in Production Example 6 except that the 1430R film was previously subjected to plasma treatment. This cup is referred to as a “surface hydrophilized 1430R cup”.

Example 9
To a cup made of 1430R, a medium [a Ham medium (manufactured by Nakarai Co., Ltd.) plus a final concentration of 10% fetal bovine serum (FBS)) was added, and the CHO cells had a cell density of 1.25 ×. The cells were seeded at 10 ⁴ cells / cm ² , placed in a CO ₂ incubator set at a temperature of 37 ° C. and a CO ₂ concentration of 5%, and cultured for 3 weeks (repeated sample number N = 3).
After culturing for 3 weeks, the cells were collected by trypsin treatment, trypan blue (DS Pharma) was added to stain the dead cells, and a Thomas type hemocytometer (Elma, notification number 13B1X90004000005) was used. The number of viable cells was counted.

[Comparative Example 4]
In Example 9, a Pyrex (registered trademark) glass tube (a glass tube of the same size as a 1430R cup) having an inner diameter of 20 mm, a thickness of 1 mm, and a length of 18 mm is placed in each well of a polystyrene 12-well plate as a culture container. The number of viable cells was counted by culturing in the same manner as in Example 9 except that a plate containing slag (called “polystyrene plate”) was used.
As shown in FIG. 6, it can be seen that the 1430R cup has an effect of maintaining about 1.7 times the viable cells as compared to the polystyrene plate.

Example 10
In Example 9, VERO cells were used instead of CHO cells, and MEM medium (manufactured by Nacalai Co., Ltd.) was added as a medium to which bovine serum (Calf Serum (CS)) with a final concentration of 10% was added. In the same manner as in Example 9, the cells were cultured using a 1430R cup, and the number of viable cells was counted.

[Comparative Example 5]
In Example 10, the cells were cultured in the same manner as in Example 10 except that a polystyrene plate was used instead of the 1430R cup, and the number of viable cells was counted.
As shown in FIG. 7, it can be seen that the 1430R cup has an effect of maintaining about 1.7 times as many viable cells as CHO cells as well as VERO cells compared to polystyrene plates.

Example 11
After culturing CHO cells using a 1430R cup for 3 weeks in the same manner as in Example 9, the enzyme activity (LDH activity) of lactate dehydrogenase in the culture solution is measured using an LDH measurement kit (Takara Bio Inc.). The leakage from inside the cells was examined.

Example 12
In Example 11, cells were cultured and LDH activity was measured in the same manner as in Example 11 except that a surface hydrophilized 1430R cup was used instead of the 1430R cup.

[Comparative Example 6]
In Example 11, cells were cultured and LDH activity was measured in the same manner as in Example 11 except that instead of the 1430R cup, a polystyrene plate was used.

When the LDH activity in the culture medium when cultivated on a polystyrene plate for 3 weeks is 1, the LDH activity in the culture liquid when cultured for 1 week in a cup made from 1430R and a cup made from surface hydrophilized 1430R is shown. Calculated.
As shown in FIG. 8, when a surface hydrophilized 1430R cup is used, there is less leakage by about 40% than when a polystyrene plate is used, and when a 1430R cup is used, a polystyrene plate is used. There was less leakage about 60% than when it was.

Example 13
After culturing VERO cells for 3 weeks using a 1430R cup in the same manner as in Example 10, the LDH measurement kit (manufactured by Takara Bio Inc.) was used to measure the enzyme activity of LDH in the culture solution, thereby Investigated leakage from inside.
Example 14
In Example 13, cells were cultured and LDH activity was measured in the same manner as in Example 13 except that a surface hydrophilized 1430R cup was used instead of the 1430R cup.

[Comparative Example 7]
In Example 13, cells were cultured and LDH activity was measured in the same manner as in Example 13 except that polystyrene plates were used instead of 1430R cups.

When the LDH activity in the culture medium when cultivated on a polystyrene plate for 3 weeks is 1, the LDH activity in the culture liquid when cultured for 1 week in a cup made from 1430R and a cup made from surface hydrophilized 1430R is shown. Calculated.
As shown in FIG. 9, in the VERO cell, when using a surface hydrophilized 1430R cup, there is about 20% less leakage than when using a polystyrene plate, and when using a 1430R cup, Leakage was about 50% less than when polystyrene plates were used.
As described above, the effect of suppressing the leakage of the cell contents of the alicyclic structure-containing polymer molded product was confirmed regardless of the type of cells.

Example 15
In Example 2, the culture was performed in the same manner as in Example 2 except that the culture period was 2 weeks, and ERK phosphorylation was analyzed.

Example 16
In Production Example 2, a culture container was obtained in the same manner as in Production Example 2 except that γ-ray sterilization was performed instead of EOG sterilization.
Except that this culture vessel was used, the culture was carried out in the same manner as in Example 15 to analyze the phosphorylation of ERK.

Example 17
In Production Example 2, a culture vessel was obtained in the same manner as in Production Example 2 except that steam sterilization was performed instead of EOG sterilization.
Except that this culture vessel was used, the culture was carried out in the same manner as in Example 15 to analyze the phosphorylation of ERK.

[Comparative Example 8]
In Example 15, culture was performed in the same manner as in Example 15 except that a FALCON container (sterilized by γ-rays) manufactured by Becton Decktonson was used, and ERK phosphorylation was analyzed.

Analysis of ERK phosphorylation was performed in the same manner as described above.
As a relative value with the value of p44 ERK and p42 ERK when using a comparative FALCON container as 100, the values when using an EOG sterilization container, a γ-ray sterilization container, and a steam sterilization container are respectively used. FIG. 10 shows a comparison of p44 activation and FIG. 11 shows a comparison of p42 activation.
Activation of p44 ERK is higher in 1430 containers compared to FALCON containers. Moreover, when the sterilization methods are compared, it can be seen that the EOG sterilization treatment has the highest activity (FIG. 10).
Even in the activation of p42 ERK, the 1430 container is higher than the FALCON container. Moreover, when the sterilization methods are compared, it can be seen that the EOG sterilization treatment has the highest activity (FIG. 11).

According to the present invention, activation by phosphorylation of ERK or AKT, which is an intracellular signal transduction protein, can be enhanced without a special additive factor. For this reason, while killing of a cultured cell can be suppressed and a living cell can be maintained, the leakage from the inside of a cell is controlled by reducing dead cells.
By using the present invention, when cell culture is used for recombinant pharmaceutical production or the like, it is considered that cells can be maintained for a long period of time and production can be continued. Furthermore, since leakage of intracellular components is small, it is possible to reduce the process load of refining recombinant pharmaceuticals. As a result, production efficiency can be improved and it can contribute to economic improvement.
In recombinant pharmaceutical production, since leakage from the cells can be reduced, it is expected that the risk of mixing unknown components from the pharmaceutical product can be reduced, and the risk of side effects can also be reduced.
Since activation of ERK and the like can also be expected to improve the differentiation of cells, the target cells can be induced to differentiate efficiently and in a short period of time for regenerative medicine and the like.

Claims (6)

1. A method for enhancing phosphorylation of ERK (Extracellular signal-Regulated Kinase) in cultured cells, which comprises contacting a cultured cell with an alicyclic structure-containing polymer molded product.
2. A method for enhancing phosphorylation of AKT in cultured cells, which comprises contacting a cultured cell with an alicyclic structure-containing polymer molded product.
3. The method for enhancing phosphorylation according to claim 1 or 2, wherein the cultured cells are adherent cells.
4. An alicyclic structure-containing polymer molded product is brought into contact with a cultured cell, and ERK (Extracellular signal-Regulated Kinase) phosphorylation and / or AKT phosphorylation in the cultured cell is enhanced, Cell culture method.
5. An agent for enhancing phosphorylation of intracellular ERK in cultured cells, comprising an alicyclic structure-containing polymer molded product.
6. An agent for enhancing phosphorylation of intracellular AKT in cultured cells, comprising an alicyclic structure-containing polymer molded product.

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