WO2017010533A1 - Method for promoting extracellular matrix production, method for culturing cells, and agent for promoting extracellular matrix production - Google Patents

Abstract

The present invention is: a method for promoting extracellular matrix production in cultured cells, the method being characterized by bringing cells that are being cultured into contact with an alicyclic structure-containing polymer molding; a method for culturing cells, the method being characterized by bringing cells that are being cultured into contact with an alicyclic structure-containing polymer molding, and promoting extracellular matrix production by the cultured cells; and an agent for promoting extracellular matrix production in cultured cells, the agent being composed of an alicyclic structure-containing polymer molding. According to the present invention, increasing the amount of extracellular matrix produced by cells makes it possible to increase the amount of protein expressed by foreign protein-producing cells without the use of a culture vessel coated with extracellular matrix.

Description

Extracellular matrix production promotion method, cell culture method, and extracellular matrix production promoter

The present invention relates to a method for promoting production of extracellular matrix produced by cultured cells, and an extracellular matrix production promoter used therefor.

In order to maintain survival while cells undergo growth stimulation, it is necessary that receptors such as integrins existing on the membrane surface of cells bind to the extracellular matrix existing outside the cells.
To cultivate various cells such as hepatocytes, blood-related cells, cancer cells, etc., or to evaluate compound toxicity and drug efficacy, or search for new genes while culturing these cells In culture experiments such as the above, in order to enhance the effects of cell proliferation and survival maintenance, a method of coating an extracellular matrix on a surface such as a plastic container bottom for cell culture that is in contact with cells is widely used. The extracellular matrix is a protein such as collagen, fibronectin, and laminin.
For example, in a culture container coated with collagen, muscle cells, hepatocytes, spinal ganglia, embryonic lung cells, schwan cells, epithelial cells (endothelium), muscle cells, nerve cells, etc. are cultured (for example, Patent Document 1). ) In a culture vessel coated with fibronectin, epithelial cells, endothelial cells, muscle cells, nerve cells, cancer cells, and the like are cultured (for example, Patent Document 2). In a culture container coated with laminin, epithelial cells, endothelial cells, nerve cells, muscle cells, hepatocytes, and the like are cultured (for example, Patent Document 3).

Japanese Patent Laid-Open No. 5-260950 Japanese Patent Publication No. 63-026988 JP-A-8-173144

In order to use a culture vessel in which the inside of the culture vessel is coated with an extracellular matrix component, a coating reagent containing the extracellular matrix component is used, and it takes time and effort for the operation. After coating, the extracellular matrix component is applied to the vessel. It is necessary to leave the plate in a clean environment for several hours at room temperature to 37 ° C. or overnight at 2-8 ° C. until it adsorbs.
When an experimenter performs a coating operation, there is a concern that the coating state of the extracellular matrix may not be constant, which is one of the causes when the reproducibility of the experiment cannot be obtained.
It is also conceivable to purchase and use a pre-coated culture vessel in order to avoid the coating operation of the experimental operator and to reduce the coating variation.
However, in addition to the increase in container cost, the container that showed the coating state of the pre-coated culture container itself (guaranteed to be in a good coating state) is not sold, and the experiment operator actually Compared to the case where the coating operation is performed, it is not possible to reduce the coating variation and to eliminate the concern about the reproducibility deterioration due to the coating variation.

In addition, since extracellular matrix components are prepared from living tissues and used, PCR tests are performed on viruses and bacteria that are a number of different pathogens, and bacteria are tested in microbial culture tests and immunoassays. , Fungi, and mycoplasmas are confirmed to be negative for infection, and endotoxin levels must be evaluated by the Limulus Amoebocyte Lysate assay. There is a problem of increasing costs in commercial use. Furthermore, since these tests are sampling tests rather than full-scale tests, the risks in use cannot be completely avoided as a result.

Since the extracellular matrix is a protein component, the risk of losing biological and physiological activities due to the influence of the management environment such as temperature, time, vibration, and light during the production of the extracellular matrix. There is also. In addition, in order to confirm that the biological and physiological activities are not lost, a sampling test is performed, but a long-term test using cells such as a neurite outgrowth assay is performed. This is a factor that increases operational complexity.

The present invention has been made in view of the above circumstances, and by increasing the amount of extracellular matrix produced by the cells, foreign protein-producing cells can be expressed without using a culture vessel coated with the extracellular matrix. It is an object of the present invention to provide a method for promoting extracellular matrix production of cultured cells, a method for culturing cells, and an agent for promoting extracellular matrix production of cultured cells, which can increase the amount of foreign protein to be produced.

As a result of intensive studies to solve the above-mentioned problems, the present inventors can increase the amount of extracellular matrix produced by bringing the alicyclic structure-containing polymer molded body into contact with cells in culture. As a result, the present invention has been completed.

Thus, according to the present invention, there is provided a method for promoting extracellular matrix production of cultured cells, which comprises contacting cultured cells with an alicyclic structure-containing polymer molded product.
The extracellular matrix is preferably a fibrous protein, more preferably selected from the group consisting of collagen, fibronectin, elastin, and laminin, and particularly preferably laminin 511. Moreover, it is preferable that the cell cultured here is a CHO cell.
In addition, according to the present invention, there is provided a method for culturing a cell, wherein the cultured cell is brought into contact with a polymer molded body containing an alicyclic structure, and the cultured cell promotes extracellular matrix production. .
Furthermore, according to this invention, the extracellular matrix production promoter of a cultured cell which consists of an alicyclic structure containing polymer molded object is provided.

FIG. 1 is a graph comparing the expression levels of extracellular matrix. FIG. 2 is a graph comparing erythropoietin production.

The cells used in the present invention are not particularly limited and can be arbitrarily selected according to the purpose.
The cell used in the present invention may be a gene-transferred cell having a property of expressing a foreign gene by genetic manipulation. Adhesive 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 (extracellular matrix) under normal culture conditions, and are also referred to as anchorage-dependent cells.
Adherent cell-derived cells are those that do not adhere to the extracellular matrix by applying some external factor to the adherent cells, such as cells that have been cultivated and cultivated, and can survive and proliferate even in suspension. It is a viable and proliferative cell.
Examples of the adherent cells include genetically engineered host cells and virus-sensitive cells represented by CHO cells, VERO cells, NIH3T3 cells, HEK293 cells, etc. Among them, CHO cells are preferable.

In the present invention, the extracellular matrix is a substance that exists in the extracellular space, supports the living tissue, and is produced by the cells. Examples of such a substance include fibrous polysaccharides and complex polysaccharides such as proteoglycan in which a polysaccharide (glucosaminoglycan) and a protein are covalently bonded. Among these, fibrous protein is preferable because production is promoted particularly effectively by the extracellular matrix production promoter for cultured cells of the present invention.

The extracellular matrix that is a fibrous protein is known as collagen, cadherin, fibronectin, laminin, vitronectin, elastin and the like. More specifically, SPONDIN 1, SPONDIN 2, extracellular matrix protein 1, extracellular matrix protein 2, extracellular matrix protein 1, EGF-containing fibulin-like extracellular matrix protein 2, elastin microfibril interfacer 1, and elastin microfibrils interfacer such 2 Examples include elastin.
In the present invention, more preferred extracellular matrix includes collagen, fibronectin, laminin and elastin, with laminin 511 being particularly preferred.

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; gene 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, hydrodynamics method 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. %. When the ratio 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 And hydrides thereof. Examples of what can be 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 monomer is preferable from the viewpoint of heat resistance, mechanical strength, and the like. From the viewpoint of operability of cell culture, a norbornene monomer having no polar group is preferable. Ring-opening polymer hydrides are particularly preferred.

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: 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- And tetracyclic monomers such as tetrahydrofluorene and 1,4-methano-8-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, 1,5,9-cyclododecatriene, 1,5,9,13-cyclohexadecatetra And monocyclic cycloolefin monomers such as ene.
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 norbornene-based monomer is usually added to the polymerization solution of the ring-opening polymer.
It can be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to hydrogenate the carbon-carbon unsaturated bond.

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 vinylcyclohexene and vinylcyclohexane, and hydrides thereof; And hydrides of aromatic ring portions of polymers of vinyl aromatic monomers such as styrene and α-methylstyrene. The vinyl alicyclic hydrocarbon polymer may be a copolymer of these monomers and other monomers copolymerizable therewith.

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, and optical brighteners can be added.

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 of the alicyclic structure-containing polymerization and the compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Moreover, there is no special restriction | limiting in 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 extracellular matrix production promoter for cultured cells 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 addition, the surface of these molded bodies can be subjected to treatments generally applied to culture vessels such as plasma treatment, corona discharge treatment, ozone treatment, ultraviolet irradiation treatment, etc., but in terms of the rate of phosphorylation enhancement of ERK and AKT. Therefore, it is preferable to use without performing these treatments.
Furthermore, 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.

The method of bringing the cultured cell into contact with the alicyclic structure-containing polymer molded body that is the extracellular matrix production promoter of the cultured cell of the present invention may be any method depending on the shape of the extracellular matrix production promoter of the cultured cell. Adopt it. For example, a method of culturing cells in a medium mixed with an alicyclic structure-containing polymer molded body that is an extracellular matrix production promoter for cultured cells; cells in a culture vessel molded using the alicyclic structure-containing polymer A method of culturing using a culture device molded using an alicyclic structure-containing polymer, and the like, and these can also be combined.
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 dish”.

[Example 1]
2 ml of medium was put into the 790R dish obtained in Production Example 1, CHO cells were seeded at a cell density of 1.25 × 10 ⁴ cells / cm ² , and a CO ₂ incubator set at a temperature of 37 ° C. and a CO ₂ concentration of 5%. After culturing for 8 days, the gene expression level of the extracellular matrix was analyzed by the method described later.

[Comparative Example 1]
Example 1 except that the culture vessel was replaced with the polystyrene dish [Falcon (registered trademark) dish (Becton Dickinson, Model 353001)] instead of the 790R dish obtained in Production Example 1. Culture was performed in the same manner, and the amount of extracellular matrix expression was analyzed.

<Analysis of gene expression level in extracellular matrix>
Total RNA was extracted and recovered from cells cultured in a 790R dish and cells cultured in a polystyrene dish. From the total RNA, mRNA was recovered using a poly-T oligomer, and cDNA was prepared using reverse transcriptase using the obtained mRNA as a template. Using about 20 million cDNA fragments fragmented by sonication as individual templates, DNA sequence analysis of about 100 bases in length was performed to obtain DNA sequence data.
Based on the obtained DNA sequence information, a gene mapping process is performed to obtain the number of reads that is the number of reads of the DNA sequence for a specific gene, and the genes of the extracellular matrix are used with cells cultured in a 790R dish. The number of leads analyzed was compared with the number of leads analyzed using cells cultured in polystyrene dishes.
FIG. 1 shows the relative value of the extracellular matrix expression level when a 790R dish is used, where the expression level of each extracellular matrix is 1 when a polystyrene dish is used.

From this result, it was found that the cell expression in the 790R dish increased the gene expression level of the extracellular matrix.

[Production Example 2] Preparation of EPO-producing recombinant CHO cells Plasmid pLXRN-EPO was constructed by inserting the EPO gene sequence into the expression gene insertion site of vector pLXRN (manufactured by Chlontech) containing a gene resistant to antibiotic G418. did. The EPO gene in the constructed plasmid was confirmed by analyzing its base sequence.
Next, virus particles containing the EPO gene were prepared by transducing the constructed plasmid pLXRN-EPO and the plasmid pVSV-G (Clontech) into package cells GP293T cells (Clontech).
In addition, Lipofectamine (manufactured by Invitrogen) was used as a gene introduction reagent for the introduction of plasmid pLXRN-EPO into cells, and the gene introduction was performed according to the manufacturer's manual.

The GP293T cells subjected to the gene transfer operation are cultured, the culture supernatant is removed, filtered, and 8 μg / ml polybrene (manufactured by Santacruz) is added to the culture containing virus particles retaining the EPO gene. A clear sample was prepared.
Subsequently, by removing the culture medium of the CHO cell sample cultured in advance and adding the culture supernatant sample containing virus particles retaining the above EPO gene and maintaining the culture for 8 hours, the virus retaining the EPO gene The particles were infected with CHO cells.
After 8 hours of incubation, the culture medium for CHO cells was changed to a culture operation for CHO cells producing recombinant EPO.

Viral infection was confirmed by genomic PCR as follows. First, using Instagene (manufactured by BioRad), the genome was extracted from the infected CHO cells, and it was confirmed by PCR that the pLXRN-EPO sequence was introduced into the genome. Primers used for PCR were pLXRN-seq-F (5′-CGCCCTCCGTCTGAATTTT) and pLXRN-seq-R (TCCCTATGCAAAAGCGAAAC).
In order to select CHO cells infected with the virus and having the EPO gene incorporated in the genome, antibiotic G418 is added to the medium, maintained in culture, and drug selection is made for antibiotic G418-resistant CHO cells. Recombinant CHO cells with the ability were selected.

[Example 2]
Using the 790R dish obtained in Production Example 1 as the culture container and Ham medium containing 10% fetal calf serum as the liquid medium, the recombinant CHO cells selected in Production Example 2 are 1.25 × 10 6. ^The cells were seeded at ⁴ cells / cm ² and cultured for 17 days under a condition of 37 ° C. in a 5% CO ₂ atmosphere. At 11 days in the middle of the culture, the volume of the medium liquid volume due to the transpiration of water was reduced during the culture. Therefore, in order to maintain the medium liquid volume, a medium having the same volume as the liquid volume decreased by the transpiration was added. Using the medium on the 17th day of culture, the amount of active EPO was measured by ELISA (human EPO Platinum ELISA manufactured by eBioscience) to determine the amount of EPO produced per unit culture area.

[Comparative Examples 2 to 7]
In Example 1, instead of the 790R dish, a polystyrene dish (Falcon registered trademark) dish (Becton Dickinson, Model No. 353001)), and polystyrene dish, gelatin, fibronectin, collagen 1, collagen 4, Dish coated with laminin and poly-D-lysine (manufactured by Becton Dickinson, Biocoat Dish Gelatin coated product: Product No. 345652, Fibronectin coated product: Product No. 354402, Collagen I coated product: Product No. 354400, Collagen IV coated product: Product No. 354428 , Laminin coated product: product number 354404, poly-D-lysine (PDL) coated product: product number 354413). It was measured quantity to obtain the EPO production per unit culture area.
The results of Example 2 and Comparative Examples 2 to 7 are shown in FIG.

From this result, it was found that the expression level of erythropoietin increases when cultured in a 790R dish compared to a polystyrene dish or a dish in which various extracellular matrices are coated on a polystyrene dish.

Claims (7)

A method for promoting extracellular matrix production of cultured cells, which comprises contacting a cultured cell with an alicyclic structure-containing polymer molded product. The method for promoting extracellular matrix production of cultured cells according to claim 1, wherein the extracellular matrix is a fibrous protein. The method for promoting extracellular matrix production of cultured cells according to claim 1, wherein the extracellular matrix is one or more selected from the group consisting of collagen, fibronectin, elastin, and laminin. The method for promoting extracellular matrix production of cultured cells according to claim 1, wherein the extracellular matrix is laminin 511. The method for promoting extracellular matrix production of cultured cells according to any one of claims 1 to 4, wherein the cultured cells are CHO cells. A method for culturing a cell, wherein the cultured cell is brought into contact with a polymer molded product containing an alicyclic structure, and the cultured cell promotes extracellular matrix production. An extracellular matrix production promoter for cultured cells, comprising an alicyclic structure-containing polymer molded product.

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