WO2015199118A1 - Procédé pour favoriser la phosphorylation d'erk ou d'akt dans une cellule cultivée, procédé de culture cellulaire et promoteur de phosphorylation - Google Patents

Procédé pour favoriser la phosphorylation d'erk ou d'akt dans une cellule cultivée, procédé de culture cellulaire et promoteur de phosphorylation Download PDF

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WO2015199118A1
WO2015199118A1 PCT/JP2015/068154 JP2015068154W WO2015199118A1 WO 2015199118 A1 WO2015199118 A1 WO 2015199118A1 JP 2015068154 W JP2015068154 W JP 2015068154W WO 2015199118 A1 WO2015199118 A1 WO 2015199118A1
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phosphorylation
cells
erk
container
cell
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PCT/JP2015/068154
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Japanese (ja)
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直也 市村
孝明 平野
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日本ゼオン株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology

Definitions

  • 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.
  • ERK Extracellular signal-Regulated Kinase
  • AKT AKT
  • 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.
  • ERK or AKT intracellular signal transduction system protein
  • integrins phosphorylation of intracellular signal transduction system protein called ERK or AKT in cells via integrins
  • 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.
  • 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.
  • 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. .
  • the product made from polystyrene is used widely as a commercial product.
  • 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 ⁇ -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.
  • 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
  • phosphorylation enhancing methods (1) to (3), (4) cell culturing methods, and (5) and (6) phosphorylation enhancing agents.
  • 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.
  • a method for enhancing phosphorylation of AKT in cultured cells which comprises contacting a cultured cell with an alicyclic structure-containing polymer molded product.
  • 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. 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
  • 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.
  • 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.
  • 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.
  • a liquid medium When culturing cells, a liquid medium is usually used.
  • 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.
  • 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.
  • aqueous solution examples 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.
  • the nutrient component of the cell include amino acids, nucleic acids, vitamins, minerals and the like.
  • various commercial products such as RPMI-1640, HAM, ⁇ -MEM, DMEM, EMEM, F-12, F-10, and M-199 can be used.
  • An additive can also be mix
  • 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
  • the cell culture conditions are not particularly limited, and can be appropriately determined according to the cells to be used and the purpose.
  • 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.
  • alicyclic structure-containing polymer examples 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).
  • norbornene-based polymers and hydrides thereof are preferable from the viewpoints of heat resistance, mechanical strength, and the like.
  • 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.
  • a ring-opening polymer hydride of a norbornene-based monomer is preferable from the viewpoint of heat resistance, mechanical strength, and the like.
  • norbornene monomer examples 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 .
  • 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 .
  • 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;
  • 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.
  • ⁇ -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.
  • ⁇ -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.
  • 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.
  • a catalyst composed of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.
  • Monocyclic cyclic olefin polymer for example, an addition polymer of a monocyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used. it can.
  • 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.
  • Vinyl alicyclic hydrocarbon polymer examples 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 ⁇ -methylstyrene.
  • the vinyl alicyclic hydrocarbon polymer may be a copolymer with other monomers copolymerizable with these monomers.
  • an alicyclic structure containing polymer Although there is no special restriction
  • 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.
  • alicyclic structure-containing polymers can be used alone or in combination of two or more.
  • 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.
  • 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
  • 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.
  • a biaxial kneader 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.
  • 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.
  • 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.
  • a plate shape, a powder form, a granular form, a string form, a sheet form, and other shapes may be sufficient.
  • 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.
  • 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.
  • the molded body when the molded body is brought into contact with the cultured cells.
  • heating methods such as the high-pressure steam method and dry heat method; radiation methods that irradiate radiation such as ⁇ rays and electron beams; irradiation methods that irradiate high frequencies; ethylene oxide gas (EOG)
  • EOG ethylene oxide gas
  • 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.
  • 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.
  • any method may be adopted depending on the shape of the ERK or AKT phosphorylation enhancer. That's fine.
  • 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.
  • the cells 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.
  • 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 5 a culture vessel was obtained in the same manner as in Production Example 4 except that 1430R was used instead of 790R.
  • 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.
  • 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.
  • polystyrene dish Falcon (registered trademark) dish (Becton Dickinson, model number 353001)
  • 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.
  • 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.
  • 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).
  • Buffer solution TBS-T Tris phosphate buffer solution
  • Tween registered trademark
  • 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.
  • 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.
  • the value obtained by dividing the reaction signal value of anti-phosphorylated ERK antibody by the reaction signal value of anti-ERK antibody 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.
  • 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.
  • 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.
  • Example 5 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.
  • 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.
  • 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.
  • 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.
  • Example 7 it culture
  • 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.
  • 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 ⁇ -ray sterilization to obtain a culture cup. This cup is referred to as a “1430R cup”.
  • 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”.
  • 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.
  • 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.
  • polystyrene plate a plate containing slag
  • 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.
  • MEM medium manufactured by Nacalai Co., Ltd.
  • bovine serum Calf Serum (CS)
  • 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.
  • LDH activity enzyme 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 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.
  • Example 6 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.
  • 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.
  • Example 7 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.
  • Example 15 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 ⁇ -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.
  • Example 15 culture was performed in the same manner as in Example 15 except that a FALCON container (sterilized by ⁇ -rays) manufactured by Becton Decktonson was used, and ERK phosphorylation was analyzed.
  • FIG. 10 shows a comparison of p44 activation
  • FIG. 11 shows a comparison of p42 activation.
  • Activation of p44 ERK is higher in 1430 containers compared to FALCON containers.
  • the sterilization methods are compared, it can be seen that the EOG sterilization treatment has the highest activity (FIG. 10).
  • the 1430 container is higher than the FALCON container.
  • the sterilization methods are compared, it can be seen that the EOG sterilization treatment has the highest activity (FIG. 11).
  • ERK or AKT which is an intracellular signal transduction protein
  • ERK or AKT which is an intracellular signal transduction protein
  • the leakage from the inside of a cell is controlled by reducing dead cells.
  • 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.
  • 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.

Abstract

L'invention concerne : un procédé pour favoriser la phosphorylation d'une kinase régulée par un signal extracellulaire (ERK) ou d'une AKT dans une cellule cultivée; un procédé de culture cellulaire utilisant le procédé pour favoriser la phosphorylation; et un promoteur de phosphorylation utilisable de manière appropriée dans ces procédés. La phosphorylation d'une ERK ou d'une AKT dans une cellule cultivée peut être favorisée par la mise en contact de la cellule cultivée avec un moulage d'un polymère contenant une structure alicyclique.
PCT/JP2015/068154 2014-06-26 2015-06-24 Procédé pour favoriser la phosphorylation d'erk ou d'akt dans une cellule cultivée, procédé de culture cellulaire et promoteur de phosphorylation WO2015199118A1 (fr)

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WO2017115706A1 (fr) * 2015-12-28 2017-07-06 日本ゼオン株式会社 Procédé pour favoriser la différenciation de cellules cultivées et agent favorisant la différenciation de cellules cultivées

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