JP2008194003A - Culture vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture vessel in which a gene transfer agent insoluble in water at a relative high temperature is deposited to the inner surface. <P>SOLUTION: The culture vessel in which the gene transfer agent containing a nucleic acid is deposited to the inner surface of the vessel is provided. The gene transfer agent is hydrophilic at a lower temperature than a prescribed temperature (T) which is lower than a living body temperature and hydrophobic at a higher temperature than the prescribed temperature (T). The gene transfer agent is obtained by preferably using a compound having three or more N,N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule as an iniferter, subjecting compound to light irradiation living polymerization of a vinylic monomer and carrying out block polymerization of the resultant homopolymer composed of the branched type polymer with N-isopropylacrylamide or a derivative thereof. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a culture vessel such as a culture plate, a petri dish or a flask used for culturing cells / microorganisms when conducting a genetics experiment.

I. JP-A-2004-290111 has a hydrophilic layer having a contact angle with water of 10 degrees or less on the inner surface of a container in contact with a culture solution as a culture container for conducting a genetics experiment. A cell culture vessel having a culture bed suitable for cell culture at a portion where the cells contact is described.
II. In recent years, gene therapy research has become more and more important as the molecular genetic factors of human diseases become clear. Gene therapy is aimed at the expression of DNA at the target site, how to make the DNA reach the target site, how to efficiently introduce the DNA into the target site and make it functionally expressed at the site It becomes important. As vectors for the introduction of foreign DNA, many viruses, including retroviruses, adenoviruses or herpes viruses, have been modified to carry therapeutic genes and are used in human clinical trials for gene therapy. However, the risk of infection and immune response remains.

As a non-viral vector for transporting DNA into cells, a cationic star polymer is described in WO2004 / 092388.
JP-A-2004-290111 WO2004 / 092388

  In the above WO2004 / 092388, since the nucleic acid complex of the nucleic acid and the gene introduction agent is dispersed in the aqueous solution, even if it is attached to the inner surface of the culture vessel, it is eluted when water is supplied to the culture vessel.

  An object of the present invention is to provide a culture vessel in which a gene introduction agent that is insoluble in water having a relatively high temperature is attached to the inner surface.

  The culture container of the present invention is a culture container in which a gene introduction agent is attached to the inner surface of a container in contact with a culture solution, the gene introduction agent comprising a polymer material having a branched chain and a nucleic acid, It is characterized in that it is hydrophilic at a temperature lower than a predetermined temperature (T) lower than the living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T). The polymer referred to in the present invention includes oligomers such as monomer dimers.

  The polymer material is preferably a polymer having a copolymer block of at least a cationic monomer and N-isopropylacrylamide or a derivative thereof, and includes a cationic monomer, a nonionic monomer, and N-isopropylacrylamide or a derivative thereof. A copolymer may also be used.

  The molecular weight of the cationic polymer block is preferably 2,000 to 500,000, and the molecular weight of the nonionic polymer block is preferably 2,000 to 500,000.

  The molecular weight of this block copolymer is preferably 3,000 to 600,000.

  The predetermined temperature (T) is preferably a temperature between 25 and 35 ° C.

  The cationic polymer is composed of a branched polymer in which a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule is used as an iniferter and a vinyl monomer is subjected to light irradiation living polymerization. A cationic homopolymer is preferably obtained by block copolymerization of N-isopropylacrylamide or a derivative thereof or a nonionic monomer.

  The compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule has a benzene ring as a nucleus and three or more N, N-dialkyl-dithio as a branched chain in this nucleus. Those having a carbamylmethyl molecular group bonded thereto are preferred.

  The vinyl monomer is at least selected from the group consisting of 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide, and methoxyethyl (meth) acrylate. One is preferred.

  In the present invention, the gene introduction agent attached to the inner surface of the culture vessel is hydrophilic and water-soluble at a temperature lower than the above-mentioned predetermined temperature. By adhering, and then setting the temperature higher than a predetermined temperature, it becomes hydrophobic (water insoluble) and adheres to the inner surface of the container.

  In the present invention, the complex of the gene introduction agent and the nucleic acid may be attached to the inner surface of the container and then dried. By drying, it is possible to expect maintenance of sterility and improvement in quality stability when transporting and storing the culture container.

  When the temperature of the liquid supplied into the culture container is higher than a predetermined temperature, the gene introduction agent becomes water-insoluble, and thus the gene introduction agent does not elute in the solution. The same applies when the gene introduction agent is dried.

  Since this gene introduction agent does not use an organic solvent, it is possible to eliminate the influence of the organic solvent when culturing cells and microorganisms.

[Gene transfer agent]
The gene introduction agent attached to the inner surface of the container in the present invention is hydrophilic at a temperature lower than a predetermined temperature (T) lower than the living body temperature, and is hydrophobic at a temperature higher than the predetermined temperature (T). It consists of a temperature sensitive polymer material with branched chains.

  As the polymer material, a branched polymer in which a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule is used as an iniferter, and a vinyl monomer is subjected to light irradiation living polymerization on this compound is used. Those obtained by block copolymerization of a temperature-sensitive polymer chain with the cationic homopolymer to be obtained, or those obtained by further block copolymerization of a nonionic polymer chain are preferred.

  In this specification, the iniferter is a polymerization initiator that generates radicals upon irradiation with light, a function as a chain transfer agent, a function that bonds with the growth terminal to stop the growth, and a polymerization when light irradiation stops. It is a molecule that functions as a polymerization initiator / termination agent for stopping.

  As a compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule, three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups are bonded as a branched chain to the benzene ring. These are preferred, and specific examples are as follows. That is, as a 3-branched chain, 1,3,5-tri (bromomethyl) benzene and sodium N, N-dithiocarbamate (sodium N, N-dithiocarbamate) can be obtained by addition reaction in ethanol. , 3,5-tri (N, N-dithiocarbamylmethyl) benzene, and four branched chains include 1,2,4,5-tetrakis (bromomethyl) benzene and sodium N, N-dithiocarbamylate ( 1,2,4,5-tetrakis (N, N-dithiocarbamylmethyl) benzene obtained by addition reaction with sodium N, N-dithiocarbamate) in ethanol. Hexakis (N, N-dithio) obtained by addition reaction of bromomethyl) benzene and sodium N, N-dithiocarbamate in ethanol Rubamirumechiru) is a benzene.

  The above iniferter is almost insoluble in polar solvents such as alcohol, but is easily soluble in nonpolar solvents. The nonpolar solvent is preferably a hydrocarbon, an alkyl halide or a halogenated alkylene, particularly benzene, toluene, chloroform or methylene chloride, and particularly preferably toluene.

As the cationic monomer to be polymerized to this iniferter, vinyl monomers such as vinyl monomers, acrylic acid derivatives, styrene derivatives and the like are particularly suitable. Specifically, 3-N, N-dimethylaminopropylacrylamide CH ₂ ═CHCONHC ₃ H ₆ N (CH ₃ ) ₂ is preferred.

  In order to cause the iniferter to react with the monomer, a raw material solution containing the iniferter and the monomer is prepared, and a reaction product in which the monomer is bonded to the iniferter is generated by irradiating the material solution with light.

  The concentration of the monomer in the raw material solution is preferably 0.5M or more, for example, 0.5 to 2.5M.

  The concentration of the iniferter is preferably about 0.1 to 100 mM.

The wavelength of the irradiated light is preferably 300 to 400 nm. The irradiation time of the light depends on the irradiation intensity, about 1 to 60 minutes are preferred, about 1 to 30 minutes at a low irradiation intensity of about 1μW ^/ cm ² ~10mW ^/ cm 2 is particularly preferred.

  In addition, you may perform a 2nd light irradiation process further after this light irradiation process (1st light irradiation process). That is, the solution containing the reaction product is diluted with an alcohol, preferably an alcohol solution of the above monomer. As the alcohol, methanol or ethanol, particularly methanol is preferable. The monomer concentration in the alcohol solution is preferably about 100 mM to 5 M as the final concentration.

  It is preferable to add 5 to 500 parts by volume of the alcohol solution to 1 part by volume of the reaction product-containing liquid from the first light irradiation step.

  The diluted solution thus diluted with the alcohol solution is subjected to the second light irradiation step, and the monomer is further polymerized with respect to the reaction product. In this case, any irradiation light source may be used as long as it includes light having a wavelength of 240 to 400 nm. For example, a low-pressure mercury lamp or a high-pressure mercury lamp can be used. The light irradiation time is preferably about 10 minutes to 120 minutes.

  By this light irradiation, a branched polymer is produced in the reaction solution, and thus a cationic homopolymer comprising the branched polymer is obtained by purification as necessary. Nonionic polymer blocks can also be obtained in a similar manner.

  The molecular weight of the cationic homopolymer block or nonionic polymer block made of this branched polymer depends on the number of branched chains, but any of them is preferably about 2,000 to 500,000, particularly 2,000 to 150,000, and particularly 2,000 to 100,000.

  N-isopropylacrylamide or a derivative thereof is block-copolymerized with the cationic homopolymer made of the branched polymer thus produced to obtain a target temperature-sensitive polymer material. This polymer block of N-isopropylacrylamide or a derivative thereof has a temperature dependency that becomes hydrophilic at a low temperature and hydrophobic at a high temperature, so that the polymer material has the temperature responsiveness.

In order to block copolymerize N-isopropylacrylamide or a derivative thereof, a cationic homopolymer composed of the branched polymer synthesized as described above is dissolved in a solvent such as methanol, and then N-isopropylacrylamide or a derivative thereof is dissolved therein. May be mixed and polymerized by irradiation with light. The concentration of the branched polymer in the solution when starting the polymerization reaction is preferably about 0.01 to 10% by weight, and the concentration of N-isopropylacrylamide or a derivative thereof is preferably about 0.3 to 30% by weight. It is. The light irradiation conditions are preferably a light wavelength of 250 to 400 nm, an irradiation time of 1 to 150 minutes, and an irradiation intensity of about 100 to 10,000 μW / cm ² .

  The molecular weight of the block copolymer (polymer material) is preferably 3,000 to 600,000, particularly preferably 3,000 to 150,000.

  In another aspect of the present invention, N-isopropylacrylamide or a derivative thereof is first polymerized with respect to the iniferter to form a homopolymer, and then a cationic monomer such as the vinyl monomer is blocked in the homopolymer. Polymerization agents may be obtained by polymerization.

  In yet another embodiment of the present invention, a cationic monomer, N-isopropylacrylamide or a derivative thereof, and a nonionic monomer are used as monomers. The order of polymerization with respect to the iniferter is arbitrary. That is, the arrangement order of the cationic polymer block, the polymer block of N-isopropylacrylamide or its derivative, and the nonionic polymer block constituting one branched chain is arbitrary.

  As the nonionic monomer, polyethylene glycol (meth) acrylate, polyethylene glycol alkyl ester (meth) acrylate, polyvinylpyrrolidone, or the like can be used.

  The gene transfer agent comprising this block copolymer is water-insoluble at a temperature higher than about 30 ° C. and water-soluble at a temperature lower than about 30 ° C.

  Therefore, an aqueous solution of a gene introduction agent complexed with nucleic acid (concentration is preferably about 3 to 150 mg / mL) at a temperature lower than about 30 ° C., for example, about 10 to 25 ° C. Let it be a culture vessel. When an aqueous liquid having a temperature higher than 30 ° C. is supplied to the culture vessel, the gene introduction agent on the inner surface of the vessel becomes water-insoluble. Therefore, the nucleic acid complex gene introduction agent is placed on the inner surface of the container for a long time, and the nucleic acid is supplied to the liquid in the culture container for a long time.

  In the present invention, a gene introduction agent (vector) made of a cationic polymer surrounds a nucleic acid as a nucleic acid-containing complex, thereby suppressing inactivation and degradation of the nucleic acid by an enzyme in a living body.

  In order to combine the cationic polymer and the nucleic acid, the nucleic acid may be added and mixed at room temperature with respect to the dispersion of the cationic polymer having a concentration of about 1 to 1000 μg / mL. It is preferable to add an excessive amount of a cationic polymer to the nucleic acid and to complex the cationic polymer in a saturated state with respect to the nucleic acid as a nucleic acid-containing complex.

[Nucleic acid]
The nucleic acid used in the present invention may be any nucleic acid that can be used in an InVitro system in fields such as cell engineering, genetic engineering, and molecular biology. Preferred examples of the nucleic acid include DNA such as a plasmid encoding luciferase, a plasmid encoding β-galactosidase (LacZ), a plasmid encoding green fluorescent protein (GFP), and various siRNAs widely used in reporter gene assays. .

  Such a series of genes is well known to those skilled in the art.

  The particle size of the nucleic acid-containing complex is preferably about 50 to 400 nm. When smaller than this, there exists a possibility that the effect | action of an enzyme may reach to the nucleic acid inside a nucleic acid containing complex.

  The nucleic acid is used in such a form that when introduced into a cell, the function can be expressed in the cell. For example, in the case of DNA, it is used as a plasmid in which the DNA is placed so that the DNA is transcribed in the introduced cell and functionally expressed through production of the polypeptide encoded thereby. Preferably, a promoter region, a start codon, DNA encoding a protein having a desired function, a stop codon, and a terminator region are sequentially arranged. Alternatively, it is useful in the present invention to perform not only gene expression but also RNA interference that destroys intracellular mRNA with siRNA.

  If desired, two or more nucleic acids can be included in one plasmid.

[container]
Examples of the container include a petri dish, a flask, a culture plate (multiwell plate), and the like. The material may be any of glass, metal, synthetic resin and the like.

[Method of using porous body of the present invention]
The adhesion amount of the gene introduction agent in the culture vessel is preferably about 0.5 ng / mm ^{2 to} 10 μg / mm ² .

  In order to attach this gene introduction agent, after pipetting, the container is gently tumbled up and down, left and right, cast and applied to the entire inner surface of the container, and then incubated at 30 to 40 ° C. for about 10 to 100 minutes. Alternatively, it may be dried as it is, preferably in a sterile atmosphere, after or without incubation.

  Moreover, when attaching, it is necessary to use properly depending on the embodiment of the present invention. For example, in the case of a 24-well or 96-well culture vessel, since it is highly possible to change the conditions for each well, it is preferable to uniformly adhere to the whole well. For example, when the gene introduction agent is attached to only one side of a 100 mm dish, it is possible to culture the gene-introduced cell and the non-gene-introduced cell in the same culture vessel.

Example 1
<Synthesis of a gene transfer agent comprising a temperature-sensitive block copolymer>
i) Synthesis of iniferter According to the following reaction formula, 1,2,4,5-tetrakis (NN-diethyldithiocarbamylmethyl) benzene was synthesized as follows.

  1.0 g of 1,2,4,5-tetrakis (bromomethylbenzene) and 4.0 g of sodium N, N-diethyldithiocarbamate were added to 100 mL of ethanol and stirred at room temperature for 4 days under light shielding. The precipitate was filtered, dried under reduced pressure, dissolved in 200 mL of chloroform, 150 mL of water was added, liquid-liquid extraction separation was performed, and sodium bromide was removed. After repeating this operation three times, the chloroform layer was dried over magnesium sulfate for 24 hours, filtered, added with n-hexane, recrystallized and purified, and white 1,2,4,5-tetrasakis ( Acicular crystals of (N, N-diethyldithiocarbamylmethyl) benzene were obtained (yield 90%). High-performance liquid chromatography confirmed that the raw material peak disappeared and the purified product was a single substance.

The measurement result of ¹ H NMR (in CDCl ₃ ) is δ1.26-1.31 ppm (t, 24H, CH ₂ CH ₃ ), δ 3.69-3.77 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), Δ 3.99-4.07 ppm (q, 8H, N (CH ₂ CH ₃ ) ₂ ), δ 4.57 ppm (s, 8H, Ar—CH ₂ ), δ 7.49 ppm (s, 2H, Ar—H) It became.

ii) Synthesis of cationic homopolymer comprising 4-branched star polymer by photopolymerization According to the following reaction formula, 1,2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) ) A cationic homopolymer comprising poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter sometimes referred to as pDMAPAAm) was synthesized.

That is, 45.6 mg of 1,2,4,5-tetrasakis (N, N-diethyldithiocarbamylmethyl) benzene synthesized by i) above was dissolved in 20 mL of chloroform, and 3-N, N-dimethylaminopropylacrylamide was dissolved. (3-N, N-DMAPAAm) 5.0 g was added and mixed, and the total amount was adjusted to 50 mL with chloroform. After purging with high-purity nitrogen gas for 5 minutes with vigorous stirring in a quartz cell, ultraviolet light was irradiated for 20 minutes with a 200 W high-pressure mercury lamp. The irradiation intensity was adjusted to 1 mW / cm ² (250 nm) using an illuminometer (UVR-1, TOPCON, Tokyo, Japan). The polymerization solution is concentrated by an evaporator, and the polymer is purified by reprecipitation with diethyl ether, dissolved in a small amount of water, filtered through a 0.2 μm filter, and lyophilized to obtain a 4-branched star homopolymer 1, A cationic homopolymer composed of 2,4,5-tetrakis [(N, N-diethyldithiocarbamyl) poly (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (pDMAPAAm) was obtained (polymerization rate). 40%). The molecular weight was determined to be 16,000 by GPC.

The measurement result of ¹ H NMR (in D ₂ O) is as follows: δ1.5-1.8 ppm (br, 2H, —CH ₂ CH ₂ CH ₂ —), δ 2.1-2.2 ppm (br, 6H, N— _{CH 3), δ2.2-2.4ppm (br,} 2H, CH 2 -N), δ3.0-3.4ppm (br, 2H, NH-CH 2), δ7.4-7.8ppm (br, 1H, -NH-).

iii) Synthesis of temperature-sensitive polymer material (4-branched pDMAPAAm-b-pNIPAM) by block copolymerization of N-isopropylacrylamide with cationic homopolymer According to the following reaction formula, 1, 2, 4 , 5-Tetrakis [(N, N-diethyldithiocarbamyl) -poly- (N-isopropylacrylamide) -block-poly- (3-N, N-dimethylaminopropylacrylamide) -methyl] benzene (hereinafter, pDMAPAAm- b-pNIPAM))).

That is, the 4-branched pDMAPAAm homopolymer synthesized in the above ii) was transferred to a 1 liter flask, re-precipitated by adding about 800 mL of diethyl ether, and the diethyl ether was removed by decantation. About 50 mL of toluene was added thereto to dissolve the polymer component, and diethyl ether was added again to reprecipitate the polymer component. This operation was repeated three times. The polymer component was dissolved in about 20 mL of methanol, and 0.1 g of N-isopropylacrylamide (NIPAM) was mixed to adjust the total amount to 60 mL with methanol. Photoirradiation polymerization was performed under the same conditions as in ii) to obtain a polymer material pDMAPAAm-b-pNIPAM composed of a block polymer of 4-branched pDMAPAAm and polyN-isopropylacrylamide (pNIPAM). The molecular weight was determined to be 19,000 by GPC.
The gene introduction agent was obtained by the above.

<Creation of culture plate>
The culture plate was prepared in a clean bench controlled at an environmental temperature of 20 to 25 ° C. All the solutions such as the buffer solution and physiological saline used for the preparation were also adjusted to 20 to 25 ° C. As the DNA, a plasmid (Promega, pGL3 control vector) encoding firefly luciferase was used.

  As the culture plate, as shown in FIG. 1, 36-well plates with A to F rows and 1 to 6 columns A1 to F6 were used.

  The solvent for DNA was x2TE buffer, and the 4-branched pDMAPAAm-b-NIPAM block polymer was dissolved in physiological saline.

  The solution to be coated onto row A was fixed at a DNA concentration of 6.66 ng / μL, and the concentrations of the 4-branched pDMAPAAm-b-NIPAM block polymer to be mixed were 0.025 μg / μL, 0.05 μg / μL, 0.15 μg. / ΜL, 0.25 μg / μL, 0.50 μg / μL, or 0.75 μg / μL, 90 μL of DNA solution and 6 types of 4 branched pDMAPAAm-b-NIPAM block polymer solution mixed at a ratio of 60.3 μL A polyplex solution was prepared, and 25 μL was dispensed into each of the wells A1, A2, A3, A4, A5, and A6, respectively, and uniformly spread into the wells.

  The DNA concentration of the solution to be coated on row B was fixed at 13.33 ng / μL, and for the 4-branched pDMAPAAm-b-NIPAM block polymer, 6 types of polyplex solutions were prepared in the same concentration series as row A. Similarly, each well of B1 to B6 was coated.

  In the same manner, for each of the remaining rows, the DNA concentration was fixed, and all wells of the 36-well culture plate were coated with the concentration sequence of the 4-branched pDMAPAAm-b-NIPAM block polymer. The DNA concentration in each row was fixed at 0.020 μg / μL for row C, 0.033 μg / μL for row D, 0.067 μg / μL for row E, and 0.10 μg / μL for row F.

  This plate was incubated at 37 ° C. for 30 minutes, and water was evaporated as it was to obtain a culture plate in which the gene transfer conditions of the present invention could be easily set.

A suspension of COS-1 cells was prepared at a cell density of 2 × 10 ⁴ EA / mL, and 1 mL was dispensed into each well of the 36-well plate prepared above and cultured. Similarly, three types of suspensions having a cell density of 6 × 10 ⁴ EA / mL, 10 × 10 ⁴ EA / mL, and 16 × 10 ⁴ EA / mL are prepared, and 1 mL is dispensed into each well of a separate 36-well plate. Injected and cultured. The state of the cells was confirmed at intervals of several hours, and morphological changes and the ratio of dead cells were observed.

After 48 hours of culture, a reporter gene assay was performed to evaluate gene expression in each well. As a result, high gene expression efficiency was confirmed in the D3 and D4 wells in the culture plate having a cell density of 6 × 10 ⁴ EA / mL compared to other wells, and almost no gene expression was confirmed in the first and second rows. There wasn't. In any culture plate, very strong cytotoxicity was confirmed in lines 5 and 6.

From the above results, in gene introduction into COS cells, the cell seeding density was 6 × 10 ⁴ EA / mL, the amount of DNA was about 0.5 μg per well, and the ratio of cationic polymer to DNA (C / A ratio). Or, if the P / N ratio is preferably about 5 to 10, the condition could be set in an experiment of about 3 days. A researcher conducting a gene transfer experiment as described above can know the conditions for gene transfer into cells by dispensing 1 mL each of cell suspensions having different cell densities into the culture plate of the present invention.

Comparative Example Using the 4-branched pDMAPAAm homopolymer synthesized in the synthesis process ii) of the above example, coating onto a 36-well culture plate having a concentration series as in the example, and evaluating the gene transfer activity by the same method However, with regard to gene expression efficiency, no correlation or trend was confirmed for the series of DNA concentration and C / A ratio. This is because polyplex consisting of 4-branched pDMAPAAm homopolymer with high water solubility and DNA is eluted and diffused from the culture plate to the medium even under the culture condition temperature of 37 ° C., and the conditions among the wells are not uniform. It is thought that it became unknown.

It is a top view of a culture plate.

Claims (11)

A culture vessel in which a gene introduction agent is attached to the inner surface of the vessel in contact with the culture solution,
The gene introduction agent comprises a polymer material having a branched chain and a nucleic acid,
The culture container, wherein the polymer material is hydrophilic at a temperature lower than a predetermined temperature (T) lower than a living body temperature and is hydrophobic at a temperature higher than the predetermined temperature (T).   2. The culture vessel according to claim 1, wherein the polymer material has a copolymer block of at least a cationic monomer and N-isopropylacrylamide or a derivative thereof.   The culture vessel according to claim 2, wherein the molecular weight of the cationic polymer block is 2,000 to 500,000.   4. The culture container according to claim 2, wherein the molecular weight of the polymer block of N-isopropylacrylamide or a derivative thereof is 2,000 to 500,000.   5. The polymer material according to claim 2, wherein the polymer material has a copolymer block of a cationic monomer, N-isopropylacrylamide or a derivative thereof, and a nonionic monomer, and the molecular weight of the nonionic polymer block is 5. A culture vessel characterized by being 2,000-500,000.   The culture container according to any one of claims 2 to 5, wherein the molecular weight of the block copolymer is 3,000 to 600,000.   The culture container according to any one of claims 1 to 6, wherein the predetermined temperature (T) is a temperature of 25 to 35 ° C.   8. The block copolymer according to claim 2, wherein the block copolymer is an iniferter having a compound having three or more N, N-dialkyl-dithiocarbamylmethyl groups in the same molecule. A culture vessel characterized in that N-isopropylacrylamide or a derivative thereof is block copolymerized with a cationic homopolymer made of a branched polymer obtained by light-irradiating living polymer.   9. The compound according to claim 8, wherein the compound having three or more N, N-dialkyl-dithiocarbamylmethyl molecular groups in the same molecule has a benzene ring as a nucleus and three or more N, N- A culture vessel in which dialkyl-dithiocarbamylmethyl molecular groups are bound.   The group according to claim 8 or 9, wherein the vinyl monomer is 3-N, N-dimethylaminopropylacrylamide, 2-N, N-dimethylaminoethyl methacrylate, N, N-dimethylacrylamide, and methoxyethyl (meth) acrylate. A culture vessel characterized by being at least one selected from the group consisting of:   The culture vessel according to any one of claims 1 to 10, wherein the gene introduction agent attached to the inner surface of the vessel in contact with the culture solution is dried.

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