JP2017012018A - Method for manufacturing cell culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily manufacturing a cell culture vessel having a culture surface on which a dried temperature responsive polymer can be uniformly coated, the cell culture vessel being excellent in handleability during production and use thereof.SOLUTION: The method for manufacturing a cell culture vessel according to the present invention comprises a dissolving step of dissolving a temperature-responsive polymer or a temperature-responsive polymer composition in an organic solvent to prepare a temperature-responsive polymer solution and a coating-and-drying step of applying the temperature-sensitive polymer solution to the culture surface of the cell culture vessel and drying the temperature-sensitive polymer solution.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a cell culture device.

  A temperature-responsive polymer dissolves in water at a temperature lower than a predetermined temperature (Lower Critical Solution Temperature (hereinafter also referred to as “cloud point”)), but insolubilizes and precipitates above a predetermined temperature. It is a polymer with physical properties.

  In a cell culture vessel in which a temperature-responsive polymer is applied to a culture surface, for example, at the time of culturing at a temperature higher than a cloud point such as 37 ° C., the cells adhere to the culture surface coated with the temperature-responsive polymer and proliferate. In addition, the cell culturing device is easy to perform the cell culturing operation so that the adhered cells can be detached and collected without performing the cell detachment operation such as triprin treatment by setting it below the cloud point.

  As a temperature-responsive polymer used in such a cell culture vessel, poly (2-N, N-dimethylaminoethyl methacrylate) (PDMAEMA), which is a polymer of 2-N, N-dimethylaminoethyl methacrylate (DMAEMA), is used. ) (Cloud point: about 32 ° C.). And according to this, a cell structure can be manufactured (patent document 1).

  And as a cell culture device which can make a cell structure, it has a structural unit derived from 2-N, N-dimethylaminoethyl methacrylate and a structural unit derived from methacrylic acid as in Patent Document 2. What coat | covers the polymer aqueous solution containing a polymer is known (patent document 2).

JP 2014-029919 A JP 2014-162865 A

  In the cell culture device of Patent Document 2, it was necessary to manage the temperature so that the temperature-responsive polymer aqueous solution was less than the cloud point before being applied to the cell culture device. In particular, when an aqueous solution of a temperature-responsive polymer is stored in an environment exceeding 32 ° C., such as in a summer laboratory, the temperature-responsive polymer aggregates and adheres to the wall surface of the storage container, and the concentration of the temperature-responsive polymer changes. In some cases, the temperature-responsive polymer could not be applied to the culture surface at the target concentration, and it took time and effort to manage the temperature when manufacturing the cell culture device.

In general, since the contact angle with water is adjusted to about 70 ° on the culture surface of the cell incubator so that the cells can easily adhere to each other, water is easily repelled. In addition, the culture surface of a plastic cell culture vessel is slightly convex at the center of the culture surface that should be smooth during plastic processing such as blow molding or injection molding. It has a shape. This is a phenomenon called “sink” well known to those skilled in the art of plastic molding, and it is difficult to process the plastic so that the culture surface becomes smooth.
Therefore, when the cell culturing apparatus of Patent Document 1 is produced, when a temperature-responsive polymer aqueous solution is dropped on the culture surface to form water droplets, or when a small amount of the temperature-responsive polymer aqueous solution is added to the culture surface, In some cases, the aqueous solution aggregates on the outer peripheral edge of the surface and the temperature-responsive polymer becomes non-uniform (see FIGS. 1 and 2). In order to uniformly distribute the temperature-responsive polymer on the culture surface, a specific amount or more is required. (For example, in a 24-well plate having a well area of 200 mm ² , 150 μL / well or more (see FIG. 2A)) was required to add an aqueous solution of a temperature-responsive polymer. When cells and a culture medium were added there, the culture medium was sometimes diluted to affect the properties of the cells.

  In addition, when an aqueous solution of a temperature-responsive polymer is added so that the temperature-responsive polymer is uniformly distributed on the culture surface, the aqueous solution of the temperature-responsive polymer is not less than the cloud point before using the cell incubator ( It was necessary to precipitate the temperature-responsive polymer on the culture surface by incubating at 37 ° C., for example, and it was necessary to take time before using the cell incubator.

  As described above, the cell culturing device described in Patent Document 1 is an excellent cell culturing device that facilitates cell culturing operation. However, the culture surface is uniformly coated with a dry temperature-responsive polymer. Therefore, there has been a demand for a method for easily producing a cell culture device excellent in handleability at the time of production and use, which does not require temperature control of the aqueous solution and incubating the cell culture device before use.

  Accordingly, an object of the present invention is to provide a method for easily producing a cell culture device that can uniformly coat a dried temperature-responsive polymer on the culture surface, and is excellent in handleability during production and use of the cell culture device. Is to provide.

  That is, the method for producing a cell culture device of the present invention comprises a dissolution step of preparing a temperature-responsive polymer solution by dissolving a temperature-responsive polymer or a temperature-responsive polymer composition in an organic solvent, and the above-described temperature-responsive polymer solution. An application drying step of applying to the culture surface of the cell culture vessel and drying.

  The organic solvent is preferably at least one selected from the group consisting of alcohols, ketones and esters.

  The organic solvent is at least one selected from the group consisting of a lower alcohol having 1 to 4 carbon atoms, a lower ketone having 3 to 5 carbon atoms, and an acetic acid alkyl ester having an alkyl group having 1 to 4 carbon atoms. Is preferred.

  It is preferable that the temperature-responsive polymer solution further contains a hydrophilic molecule.

The temperature-responsive polymer is a repeating unit (A) represented by the formula (I)
And the repeating unit (B) represented by the formula (II)
It is preferable that it is a temperature-responsive polymer containing.

  The temperature-responsive polymer is preferably a polymer having a homopolymer region of the repeating unit (A) and a copolymer region of the repeating unit (A) and the repeating unit (B).

  Moreover, the cell culture device of the present invention is manufactured by the above-described method for manufacturing a cell culture device.

  According to the method for producing a cell culture device of the present invention, a cell culture device that can uniformly coat a dried temperature-responsive polymer on a culture surface and has excellent handling properties during production and use of the cell culture device. A simple manufacturing method can be provided.

FIG. 1 is a photograph of a 24-well plate containing 50 μL / well (right column), 100 μL / well (middle column), and 150 μL / well (left column) aqueous solution. FIG. 2A is an enlarged view of the well containing 150 μL / well of the aqueous solution of FIG. FIG. 2B is an enlarged view of the well containing 100 μL / well of the aqueous solution of FIG. FIG. 2C is an enlarged view of a well containing 50 μL / well of the aqueous solution of FIG. FIG. 3 is an example showing the state of “◎ (excellent)” in the evaluation of the peelability of the cell structure. FIG. 4 is an example showing a state of “◯ (good)” in the evaluation of the peelability of the cell structure. FIG. 5 is an example showing a state of “Δ (normal)” in the evaluation of the peelability of the cell structure.

  Hereinafter, embodiments of the method for producing a cell culture device of the present invention will be described in detail.

(Dissolution process)
In the method for producing a cell culture device of the present embodiment, a temperature-responsive polymer or a temperature-responsive polymer composition is dissolved in an organic solvent to be referred to as a temperature-responsive polymer solution (in this specification, simply referred to as “polymer solution”). At least a dissolution step.
According to the method for producing a cell culture device of the present embodiment, a cell culture device in which the culture surface is uniformly coated with a dried temperature-responsive polymer can be easily produced.

Since the organic solvent is excellent in solubility of the temperature-responsive polymer, it does not take time and effort to control the temperature of the polymer solution below the cloud point. In addition, since the organic solvent dries quickly, the culture surface can be uniformly coated with the temperature-responsive polymer. In addition, the organic solvent easily wets the culture surface having a high contact angle with water, and even with a small amount, the culture surface can be uniformly coated.
Cell culture vessels in which the culture surface is evenly coated with a dry temperature-responsive polymer are adhered to the culture surface, regardless of whether the temperature is low or high. There is no need to manage. Furthermore, aseptic packaging and distribution are possible, and if the user opens the aseptic packaging, the user can use it without inconvenience such as incubation before use. Therefore, it is not necessary for the user to prepare a cell culture device before use, and a stable cell culture device can be used.
In the present specification, the “cloud point” of a polymer is not necessarily a strict meaning, and refers to “the temperature at which it dissolves below a predetermined temperature but becomes insoluble and precipitates above a predetermined temperature”. In addition, it refers to “the temperature at which the time required for dissolution is 10 minutes or more when the insolubilized and precipitated polymer is dissolved under a condition lower than a predetermined temperature”.

  Hereinafter, the temperature-responsive polymer and the temperature-responsive polymer composition used in the embodiment will be described in detail.

The temperature-responsive polymer and temperature-responsive polymer composition used in the cell culture device of this embodiment include (A) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) unit and an anionic monomer unit. Temperature-responsive polymer, (B) Temperature-responsive polymer comprising N-isopropylacrylamide (NIPAM) units, cationic monomer units, and anionic monomer units, (C) 2-N, N-dimethylaminoethyl methacrylate ( DMAEMA) and / or a derivative thereof, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, Polymethacrylic acid, polyphosphoric acid, sulfated polysaccharide, curdlan and Polyalginic acid and the temperature-responsive polymer composition comprising one or more anionic material selected from the group consisting of alkali metal salts and the like. Among these, (A) is preferable.
Here, as the above (A), for example, (A-1) a temperature-responsive polymer obtained by a method of polymerizing DMAEMA in the presence of water, (A-2) a polymer block mainly containing DMAEMA (polymer chain α-terminal) And a temperature-responsive polymer mainly containing DMAEMA and an anionic monomer (polymer chain ω terminal).
In this embodiment, these may be used individually by 1 type and may be used in combination of 2 or more type.

  Hereinafter, the temperature-responsive polymer (A-1) and the production method thereof will be described.

(Method for producing temperature-responsive polymer)
In the method for producing a temperature-responsive polymer (A-1), first, a mixture containing 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) is prepared (preparation step). Here, the mixture further includes a polymerization inhibitor and water.

  A commercially available product can be used as 2-N, N-dimethylaminoethyl methacrylate (DMAEMA). Examples of the polymerization inhibitor include methylhydroquinone (MEHQ), hydroquinone, p-benzoquinoline, N, N-diethylhydroxylamine, N-nitroso-N-phenylhydroxylamine (Cupperron), t-butylhydroquinone, and the like. Further, MEHQ or the like contained in commercially available DMAEMA may be used as it is. Examples of water include ultrapure water.

The weight ratio of the polymerization inhibitor to the mixture is preferably 0.01% to 1.5%, and more preferably 0.1% to 0.5%. If it is the said range, the runaway of radical polymerization reaction can be suppressed, the uncontrollable bridge | crosslinking can be reduced, and the solubility with respect to the solvent of the temperature-responsive polymer manufactured can be ensured.
The weight ratio with respect to the water mixture is preferably 1.0% to 50%, and more preferably 9.0% to 33%. If it is the said range, the reaction rate of the hydrolysis reaction of a side chain and the reaction rate of the growth reaction of the polymer chain to superpose | polymerize can be harmonized in good balance. Thereby, the temperature-responsive polymer whose ratio (copolymerization ratio) of DMAEMA in which the side chain is not hydrolyzed with respect to DMAEMA in which the side chain is hydrolyzed can be obtained.

Next, in the method for producing a temperature-responsive polymer (A-1), the mixture is irradiated with ultraviolet rays (irradiation step). Here, ultraviolet rays are irradiated in an inert atmosphere. DMAEMA undergoes radical polymerization upon irradiation with ultraviolet rays to form a polymer.
In this step, for example, after the mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, ultraviolet rays are irradiated from the outside of the vial using an ultraviolet irradiation device.

The wavelength of the ultraviolet light is preferably 210 nm to 600 nm, and more preferably 360 nm to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

Regarding the reaction conditions, the temperature conditions are preferably 15 ° C. to 50 ° C., more preferably 20 ° C. to 30 ° C. If it is set as the said range, the start reaction by a heat | fever can be suppressed and the start reaction by light irradiation can be advanced preferentially. Further, the reaction rate of the hydrolysis reaction can be balanced with respect to the reaction rate of the growth reaction of the polymer chain.
The reaction time is preferably 7 hours to 24 hours, and more preferably 17 hours to 21 hours. If it is the said range, the temperature-responsive polymer of (A-1) can be obtained with a high yield, and radical polymerization can be performed, suppressing a photodecomposition reaction and an unnecessary crosslinking reaction.

  In addition, it is preferable that it is 10 minutes-1 hour after completing preparation of a mixture in a preparation process until it starts irradiation of an ultraviolet-ray in an irradiation process.

  It takes about 10 minutes to replace the gas inside the vial to which the mixture has been added to create an inert atmosphere inside the vial. Therefore, if the time is less than 10 minutes, there is a possibility that an inert atmosphere necessary for radical polymerization cannot be obtained. Further, in the mixture, the hydrolysis reaction of DMAEMA is started before the irradiation with ultraviolet rays is started. Therefore, if the above time is longer than 1 hour, a large number of methacrylic acid that is inert to the radical polymerization reaction is generated in the mixture.

In the method for producing a temperature-responsive polymer of (A-1), since water is contained in the mixture, the radical polymerization reaction of DMAEMA and the ester bond of the side chain of poly-2-N, N-dimethylaminoethyl methacrylate (PDMAEMA) The hydrolysis reaction can be antagonized.
The product obtained by this antagonism is the repeating unit (A) represented by the formula (I)
And the repeating unit (B) represented by the formula (II)
It becomes a polymer containing.
Therefore, both the cationic functional group that the polymer has, that is, the dimethylamino group, and the anionic functional group that the polymer has, that is, the carboxyl group that is formed by hydrolyzing the ester bond of the side chain must be provided in a balanced manner. it can. And according to the manufacturing method of the temperature-responsive polymer of (A-1), there are few polymers derived from poly (2-N, N-dimethylaminoethyl methacrylate) having a cationic functional group and an anionic functional group. It can be easily manufactured in the process.

In addition, even if it is not the same manufacturing method as the manufacturing method of the temperature-responsive polymer of (A-1), if DMAEMA, a polymerization inhibitor, and water coexist in a reaction system at the time of ultraviolet irradiation, this embodiment The effect similar to the said effect of the manufacturing method of temperature-responsive polymer of this can be acquired.
For example, a mixture containing DMAEMA and a polymerization inhibitor and water are prepared separately, and then an inert gas is bubbled into the mixture and water, and then the mixture and water are mixed under an inert atmosphere and at the same time UV The method for producing a temperature-responsive polymer in which A is irradiated can also be included in the temperature-responsive polymer of (A-1).

(Temperature responsive polymer)
The temperature-responsive polymer (A-1) is produced by the production method (A-1).

The temperature-responsive polymer (A-1) includes a homopolymer region of the repeating unit (A) and the repeating unit from the viewpoint of ensuring ion balance while maintaining a cloud point at a temperature suitable for cell culture. A polymer having a copolymer region of (A) and the repeating unit (B) is preferable.
The method for producing a polymer having a homopolymer region of the repeating unit (A) and a copolymer region of the repeating unit (A) and the repeating unit (B) is, for example, polymerized by light irradiation of DMAEMA. When the number average molecular weight of the polymer exceeds a certain value (for example, when the number average molecular weight of the polymer exceeds 5,000 Da (more preferably 20,000 Da)), the above repeating unit (B) which is an anionic monomer The method of mixing and further irradiating light is mentioned.

The temperature-responsive polymer (A-1) is preferably a molecule having a number average molecular weight (Mn) of 10 kDa to 500 kDa. Further, a molecule having a ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 1.1 to 10.0.
The molecular weight of the temperature-responsive polymer (A-1) can be appropriately adjusted according to the conditions of the ultraviolet irradiation time and irradiation intensity.

  According to the temperature-responsive polymer (A-1), the cloud point can be lowered to, for example, room temperature (25 ° C.) or lower.

  In the temperature-responsive polymer (A-1), the time until the insolubilized product of the temperature-responsive polymer formed at a temperature equal to or higher than the cloud point is redissolved under room temperature (about 25 ° C.) conditions is remarkably delayed. To do. This is presumed to be because the obtained temperature-responsive polymer (A-1) has a high self-aggregation property because a cationic functional group and an anionic functional group exist in the molecule.

  Further, using the temperature-responsive polymer of (A-1), as described later, it is possible to prepare a cell culture vessel in which the culture surface is coated with this temperature-responsive polymer.

  Furthermore, according to the temperature-responsive polymer of (A-1), as will be described later, by culturing cells under appropriate culture conditions, it has a tubular (tubular) and massive (pellet-like) structure. Cell structures can be formed.

  The ratio of the number of functional groups of the cationic functional group (2-N, N-dimethylamino group) and the number of functional groups of the anionic functional group (carboxyl group) of the temperature-responsive polymer of (A-1) (C / A ratio) is preferably 0.5 to 32, and more preferably 4 to 16.

  When the C / A ratio is in the above range, the above effect of reducing the cloud point can be easily obtained. In the temperature-responsive polymer having the C / A ratio, the cationic functional group and the anionic functional group act on the intermolecular and / or intramolecular aggregation in an ionic bond in the temperature-responsive polymer, This is presumed to be a result of increasing the cohesive force of the temperature-responsive polymer.

  In addition, when the C / A ratio is in the above range, the balance between the positive charge and the negative charge in the temperature-responsive polymer can be particularly suitable, and the cytotoxicity due to the positive charge can be suppressed. It is presumed that the balance between hydrophilicity and hydrophobicity of the temperature-responsive polymer can be made particularly suitable to facilitate cell migration and orientation.

  Hereinafter, the temperature-responsive polymer (A-2) and the production method thereof will be described.

(Method for producing temperature-responsive polymer)
In the method for producing a temperature-responsive polymer (A-2), first, ultraviolet light is irradiated to a first mixture containing 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) (first polymerization step).
Here, in addition to DMAEMA, the first mixture may optionally include, for example, other monomers, solvents, and the like.
Moreover, ultraviolet rays may be irradiated in an inert atmosphere.

DMAEMA may be a commercially available product.
Examples of other monomers that can be included in the first mixture include N, N-dimethylacrylamide, acrylic acid and methacrylic acid esters having a polyethylene glycol side chain, N-isopropylacrylamide, and 3-N, N-dimethylaminopropyl. Examples include acrylamide, 2-N, N-dimethylaminoethyl methacrylamide and the like, and in particular, from the viewpoint of enabling stable adjustment of ion balance, N, N-dimethylacrylamide and polyethylene glycol side chains. Preference is given to esters of acrylic acid and methacrylic acid and N-isopropylacrylamide. These may be used alone or in combination of two or more. Here, the ratio (number of moles) of the usage amount of the other monomer to the usage amount of DMAEMA is preferably 0.001-1, and more preferably 0.01-0.5.

  Examples of the solvent include toluene, benzene, chloroform, methanol, ethanol, and the like. In particular, toluene and benzene are preferable because they are inert to the ester bond of DMAEMA. These may be used alone or in combination of two or more.

  In this step, for example, the first mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, and then irradiated with ultraviolet rays from the outside of the vial using an ultraviolet irradiation device. To do.

The ultraviolet wavelength is preferably 210 to 600 nm, and more preferably 360 to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
The irradiation intensity of ultraviolet light is preferably 0.01~50mW / cm ^2, further preferably 0.1~5mW / cm ^2. If it is the said range, a polymerization reaction can be advanced stably at a suitable speed (time), suppressing decomposition | disassembly by the cutting | disconnection of a useless chemical bond, etc.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

As temperature conditions, it is preferable that it is 10-40 degreeC, and it is still more preferable that it is 20-30 degreeC. If it is the said range, it can react at room temperature of a normal laboratory, and it becomes possible to suppress reaction by means (heating etc.) different from light.
The reaction time is preferably 10 minutes to 48 hours, and more preferably 60 minutes to 24 hours.

  In this step, DMAEMA undergoes radical polymerization by irradiation with ultraviolet rays to form a polymer (poly (2-N, N-dimethylaminoethyl methacrylate) (PDMAEMA)), which contains 2-N, N-dimethylaminoethyl methacrylate. A homopolymer block is formed. When other monomers are also used, a polymer block containing DMAEMA and other monomers is formed.

Next, in the method for producing a temperature-responsive polymer of (A-2), the number average molecular weight of the polymer (specifically, polymerized 2-N, N-dimethylaminoethyl methacrylate) in the first polymerization step is predetermined. When it becomes more than the value, an anionic monomer is added to the first mixture to prepare a second mixture (addition step).
Here, in addition to the first mixture after the first polymerization step and the anionic monomer, the second mixture is, for example, another monomer, a solvent (toluene, benzene, methanol, etc.) that can be included in the first mixture, and the like. May be included.
The anionic monomer may be added under an inert atmosphere.

Examples of the anionic monomer include acrylic acid, methacrylic acid, vinyl derivatives having a carboxyl group, a sulfonic acid group, and a phosphoric acid group in the side chain. Particularly, from the viewpoint of chemical stability, acrylic acid, methacrylic acid, and the like. Acid is preferred.
These may be used alone or in combination of two or more.

  Examples of other monomers that can be included in the second mixture include N, N-dimethylacrylamide, acrylic acid and methacrylic acid esters having a polyethylene glycol side chain, N-isopropylacrylamide, and 3-N, N-dimethylaminopropyl. Examples thereof include acrylamide and 2-N, N-dimethylaminoethyl methacrylamide, and particularly, N, N-dimethylacrylamide which is electrically neutral and hydrophilic is preferable. These may be used alone or in combination of two or more. Here, the ratio (mole) of the use amount of the other monomer to the use amount of DMAEMA is preferably 0.01 to 10, and more preferably 0.1 to 5.

  In this step, for example, the second mixture is added while keeping the inside of the vial in an inert atmosphere by flowing an inert gas through the vial.

The predetermined value of the number average molecular weight is preferably 5,000, more preferably 20,000, and particularly preferably 100,000 from the viewpoint of sufficiently obtaining the effect of reducing the cloud point.
The number average molecular weight of the polymerized PDMAEMA in the first mixture after the first polymerization step is obtained by collecting a small amount of the reaction mixture from the polymerization system at a predetermined time, and performing gel permeation chromatography (GPC) or light scattering method. It can be measured by a method well known to those skilled in the art such as (SLS).

  In this step, in addition to the homopolymer containing DMAEMA during polymerization, an anionic monomer is also included in the polymerization system, and the polymerization system in the vial is changed from the DMAEMA homopolymerization system to the co-polymerization of DMAEMA and the anionic monomer. It will change to a polymerization system.

And in the manufacturing method of the temperature-responsive polymer of (A-2), a 2nd mixture is irradiated with an ultraviolet-ray (2nd polymerization process).
Here, the ultraviolet rays may be irradiated in an inert atmosphere.

  In this step, for example, ultraviolet rays are irradiated from the outside of the vial after adding the second mixture using an ultraviolet irradiation device.

  Various conditions such as the wavelength of ultraviolet rays, the irradiation intensity of ultraviolet rays, the inert gas used, the reaction temperature, and the reaction time in the second polymerization step may be the same as those in the first polymerization step.

  In this step, DMAEMA and anionic monomer are radically polymerized by irradiation with ultraviolet rays, and DMAEMA and anionic are continuously formed in the polymer chain α-terminal of the homopolymer block containing DMAEMA formed in the first polymerization step. A copolymer block containing monomers is formed. When other monomers are used, a copolymer block containing DMAEMA, an anionic monomer, and other monomers is formed.

  As described above, a temperature-responsive polymer including a homopolymer block containing DMAEMA and a copolymer block of DMAEMA and an anionic monomer is obtained.

  In addition, in the production method of (A-2), as understood by those skilled in the art, a mixture of polymers having various molecular weights and molecular structures is formed. A homopolymer block containing DMAEMA, DMAEMA and an anionic monomer, From the viewpoint of obtaining a temperature-responsive polymer containing the copolymer block as a main component, it is preferable to perform polymerization under the same conditions throughout the first polymerization step, the addition step, and the second polymerization step.

(Temperature responsive polymer)
The temperature-responsive polymer (A-2) is produced by the production method (A-2).

The temperature-responsive polymer of (A-2) mainly contains 2-N, N-dimethylaminoethyl methacrylate, and optionally a hydrophilic monomer such as acrylic acid or methacrylic acid having dimethylacrylamide and polyethylene glycol side chains. Polymer block containing other monomer units (polymer chain α-terminal), mainly comprising 2-N, N-dimethylaminoethyl methacrylate and anionic monomer (polymer chain ω-terminal), optionally other monomer units And a copolymer block containing.
Preferably, the temperature-responsive polymer of (A-2) comprises a DMAEMA homopolymer block and a copolymer block of DMAEMA and an anionic monomer, and more preferably consists of these blocks.

  Here, as the temperature-responsive polymer of (A-2), the number average molecular weight of the polymer block at the polymer chain α-terminal (for example, DMAEMA homopolymer block) is preferably 5000 Da or more, and 20000 Da or more. Is more preferable.

The temperature-responsive polymer (A-2) is preferably a molecule having a number average molecular weight (Mn) of 10 to 500 kDa. Further, a molecule having a ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 1.1 to 10.0.
The molecular weight of the temperature-responsive polymer can be adjusted as appropriate depending on the conditions of irradiation time and irradiation intensity of ultraviolet rays.

  According to the temperature-responsive polymer (A-2), the cloud point can be lowered to, for example, room temperature (25 ° C.) or lower.

  In the temperature-responsive polymer of (A-2), the time until the insolubilized product of the temperature-responsive polymer formed at a temperature higher than the cloud point is redissolved at room temperature (about 25 ° C.) is remarkably delayed. To do. It is presumed that this is because the obtained temperature-responsive polymer has high self-aggregation properties because of the presence of a cationic functional group and an anionic functional group in the molecule.

  In particular, the temperature-responsive polymer of (A-2) has a DMAEMA homopolymer block having a high molecular weight (for example, 5000 Da or more) at the α-terminal of the polymer chain, so that the temperature-dependent globule transition of the side chain of DMAEMA Therefore, it is considered that the cloud point can be effectively reduced.

  In addition, using this temperature-responsive polymer, as described later, it is possible to prepare a cell culture vessel in which the culture surface is coated with this temperature-responsive polymer.

  Furthermore, according to the temperature-responsive polymer of (A-2), as described later, a cell structure having a massive (pellet-like) structure can be formed by culturing cells under appropriate culture conditions. it can.

  The ratio of the number of functional groups of the cationic functional group (2-N, N-dimethylamino group) and the number of functional groups of the anionic functional group (carboxyl group) of the temperature-responsive polymer of (A-2) (C / A ratio) is preferably 0.5 to 32, and more preferably 4 to 16.

  When the C / A ratio is in the above range, the above effect of reducing the cloud point can be easily obtained. In the temperature-responsive polymer having the C / A ratio, the cationic functional group and the anionic functional group act on the intermolecular and / or intramolecular aggregation in an ionic bond in the temperature-responsive polymer, This is presumed to be a result of increasing the cohesive force of the temperature-responsive polymer.

  In addition, when the C / A ratio is in the above range, the balance between the positive charge and the negative charge in the temperature-responsive polymer can be particularly suitable, and the cytotoxicity due to the positive charge can be suppressed. It is presumed that the balance between hydrophilicity and hydrophobicity of the temperature-responsive polymer can be made particularly suitable to facilitate cell migration and orientation.

  Hereinafter, the temperature-responsive polymer (B) and the production method thereof will be described.

(Method for producing temperature-responsive polymer)
The method for producing the temperature-responsive polymer (B) is also referred to as N-isopropylacrylamide (NIPAM) (hereinafter also referred to as “monomer (A)”) and a cationic monomer (hereinafter referred to as “monomer (B)”). ) And an anionic monomer (hereinafter also referred to as “monomer (C)”). Optionally, other monomers may be added to the above three types of monomers for polymerization.

  N-isopropylacrylamide (NIPAM) may be a commercially available product.

Examples of the cationic monomer include monomers having a cationic functional group. Examples of the cationic functional group include amino groups such as primary to quaternary amino groups, guanidine groups, and the like. Tertiary amino groups are preferred from the viewpoints of the properties, low cytotoxicity, sterilization stability, and strong positive chargeability.
More specifically, the cationic monomer is preferably one having high stability even when a physiologically active substance is supported or under alkaline conditions. For example, 3- (N, N-dimethylaminopropyl) -meta (A) Chrylamide, 3- (N, N-dimethylaminopropyl) -meth (a) acrylate, aminostyrene, 2- (N, N-dimethylaminoethyl) -meth (a) acrylamide, 2- (N, N -Dimethylaminoethyl) -meth (a) acrylate and the like.
Among these, 3- (N, N-dimethylaminopropyl) acrylamide is particularly preferable because it has a high positive charge strength and facilitates loading of an anionic substance. In addition, aminostyrene has a high positive charge strength, facilitates the loading of anionic substances, and the aromatic ring in the molecule interacts with the hydrophobic structure of other substances in an aqueous solution. Is preferable in order to widen the variation of the active substance. Furthermore, 2- (N, N-dimethylaminoethyl) -methacrylamide has a weak positive charge at a neutral pH, and its solubility in water is not affected by temperature, so that it is once anionic. This is preferred because it facilitates release of the substance.
These may be used alone or in combination of two or more.

Examples of the anionic monomer include monomers having an anionic functional group, and examples of the anionic functional group include a carboxylic acid group, a sulfonic acid group, a sulfuric acid group, a phosphoric acid group, and a boronic acid group. From the viewpoints of physical stability, cell affinity, and high purity, carboxylic acid groups, sulfonic acid groups, and phosphoric acid groups are preferred.
More specifically, acrylic acid, methacrylic acid, vinyl benzoic acid and the like can be mentioned, and methacrylic acid and vinyl benzoic acid are particularly preferable from the viewpoint of chemical stability and cell affinity.
These may be used alone or in combination of two or more.

Examples of the other monomer include neutral hydrophilic monomers such as dimethylacrylamide and acrylic acid or methacrylic acid having a polyethylene glycol side chain.
These may be used alone or in combination of two or more.
Other monomers can be used to adjust the balance of hydrophilicity / hydrophobicity other than electric charge, and variations can be expanded.

  Here, the usage amount of NIPAM, the usage amount of the cationic monomer, and the usage amount of other monomers in the method for producing the temperature-responsive polymer of (B) with respect to the total usage amount of the monomers (A) to (C). The ratio (mole) can be appropriately adjusted by those skilled in the art so as to obtain a desired ratio of the monomer component in consideration of the reactivity in the polymerization reaction of the monomer.

Here, radical polymerization, ionic polymerization, etc. are mentioned as a polymerization method.
As the radical polymerization, living radical polymerization is preferable, and as living radical polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization, atom transfer radical polymerization (ATRP), iniferter polymerization and the like can be mentioned, and iniferter polymerization is preferable.
As ionic polymerization, living anionic polymerization is preferred.

An example of a method for producing the temperature-responsive polymer (B) is a method using radical polymerization.
In an example of this production method, first, a first mixture containing N-isopropylacrylamide (NIPAM) is irradiated with ultraviolet rays (first polymerization step).
Here, in addition to DMAEMA, the first mixture may optionally contain, for example, other monomers, solvents, chain transfer agents, stabilizers, surfactants, and the like.
Moreover, ultraviolet rays may be irradiated in an inert atmosphere.

  In this step, for example, the first mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, and then irradiated with ultraviolet rays from the outside of the vial using an ultraviolet irradiation device. To do.

  Examples of the solvent include benzene, toluene, chloroform, methanol, water, and the like. In particular, benzene and toluene are preferable from the viewpoint of solubility and inertness to polymerization. These may be used alone or in combination of two or more.

  In this step, for example, the first mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, and then irradiated with ultraviolet rays from the outside of the vial using an ultraviolet irradiation device. To do.

The ultraviolet wavelength is preferably 210 to 600 nm, and more preferably 360 to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
The irradiation intensity of ultraviolet light is preferably 0.01~50mW / cm ^2, further preferably 0.1~5mW / cm ^2.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

As temperature conditions, it is preferable that it is 10-40 degreeC, and it is still more preferable that it is 20-30 degreeC. With the above range, it is possible to perform the polymerization reaction at room temperature in a normal laboratory, and it is also possible to control the reaction by means of heating different from the means of light irradiation. .
The reaction time is preferably 10 minutes to 48 hours, and more preferably 60 minutes to 24 hours.

  In this step, NIPAM undergoes radical polymerization by irradiation with ultraviolet rays to become a polymer (poly (N-isopropylacrylamide) (PNIPAM)), and a homopolymer block containing N-isopropylacrylamide is formed. When other monomers are also used, a polymer block containing NIPAM and other monomers is formed.

Next, in the method for producing a temperature-responsive polymer (B), a cationic monomer and an anionic monomer are added to the first mixture after the first polymerization step to prepare a second mixture (addition step).
Here, the second mixture contains, in addition to the first mixture after the first polymerization step, the cationic monomer, and the anionic monomer, for example, other monomers, a solvent, a chain transfer agent, a stabilizer, a surfactant, and the like. It's okay.
Further, the cationic monomer and the anionic monomer may be added under an inert atmosphere.

  In this step, for example, the cationic monomer and the anionic monomer are added while keeping the inside of the vial in an inert atmosphere by flowing an inert gas through the vial.

  In this step, in addition to the homopolymer containing NIPAM during polymerization, a cationic monomer and an anionic monomer are also included in the polymerization system, and the polymerization system in the vial is changed from NIPAM homopolymerization system to NIPAM and cationic polymer. It will change to the copolymerization system of a monomer and an anionic monomer.

And in the manufacturing method of the temperature-responsive polymer of (B), an ultraviolet-ray is irradiated to a 2nd mixture (2nd polymerization process).
Here, the ultraviolet rays may be irradiated in an inert atmosphere.

  In this step, for example, ultraviolet rays are irradiated from the outside of the vial after adding the cationic monomer and the anionic monomer using an ultraviolet irradiation device.

The ultraviolet wavelength is preferably 210 to 600 nm, and more preferably 360 to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
The irradiation intensity of ultraviolet light is preferably 0.01~50mW / cm ^2, further preferably 0.1~5mW / cm ^2.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

As temperature conditions, it is preferable that it is 10-40 degreeC, and it is still more preferable that it is 20-30 degreeC. With the above range, it is possible to perform the polymerization reaction at room temperature in a normal laboratory, and it is also possible to control the reaction by means of heating different from the means of light irradiation. .
The reaction time is preferably 10 minutes to 48 hours, and more preferably 60 minutes to 24 hours.

  In this step, NIPAM, the cationic monomer, and the anionic monomer are radically polymerized by irradiation with ultraviolet rays, and are continuous with the polymer chain α-terminal of the homopolymer block containing NIPAM formed in the first polymerization step. A copolymer block comprising NIPAM, a cationic monomer and an anionic monomer is formed. When other monomers are used, a polymer block containing NIPAM and other monomers and / or a copolymer block containing NIPAM, cationic monomers, anionic monomers and other monomers are formed.

  As described above, a temperature-responsive polymer including a homopolymer block containing NIPAM and a copolymer block of NIPAM, a cationic monomer, and an anionic monomer is obtained.

  In the manufacturing method of this example, it is preferable to irradiate ultraviolet rays over the first polymerization step, the addition step, and the second polymerization step from the viewpoint of realizing an efficient reaction.

Another example of the method for producing the temperature-responsive polymer of (B) is a method using radical polymerization, and N-isopropylacrylamide (NIPAM), a cationic monomer, an anionic monomer, and optionally other The mixture containing the monomer is irradiated with ultraviolet rays.
Here, the said mixture may contain a solvent, a chain transfer agent, a stabilizer, surfactant, etc., for example.
Moreover, ultraviolet rays may be irradiated in an inert atmosphere.
Other conditions may be the same as in the above-described example manufacturing method.

  Furthermore, when using iniferter polymerization, benzyl- (N, N-diethyl) dithiocarbamate may be used as an iniferter, toluene or the like may be used as a solvent, and living polymerization may be performed by irradiation with near ultraviolet rays. Here, after the polymerization with the first monomer, the block copolymer can be obtained by performing the polymerization with the second monomer through the isolation operation.

  Furthermore, in the case of using ionic polymerization, an aprotic solvent may be used as a catalyst with NaOH powder and as a solvent with a reprecipitation solvent used for purification. After the polymerization with the first monomer, a block copolymer can be obtained by performing the reprecipitation operation (the ionic species remain at the ω end even after this operation) and the polymerization with the second monomer.

(Temperature responsive polymer)
The temperature-responsive polymer (B) is produced by the production method (B).

The temperature-responsive polymer of (B) includes N-isopropylacrylamide (NIPAM) units, cationic monomer units, and anionic monomer units, and optionally other monomer units. This polymer can be manufactured by the manufacturing method of the above-mentioned example and another example.
Preferably, the temperature responsive polymer of (B) comprises mainly a polymer block (polymer chain α-terminal) comprising N-isopropylacrylamide (NIPAM) units and optionally other monomer units, and a cationic monomer. And a copolymer block containing anionic monomer units and optionally other monomer units. More preferably, the temperature-responsive polymer of (B) includes a homopolymer block of NIPAM and a copolymer block of NIPAM, a cationic monomer and an anionic monomer, and particularly preferably comprises these blocks. This polymer can be manufactured by the manufacturing method of the above-mentioned example.

For example, in the temperature-responsive polymer described in Patent Document 1, DMAEMA that imparts temperature responsiveness to the polymer is a cationic monomer that is necessary for forming a cell structure (along with an anionic monomer) at the same time. DMAEMA related to responsiveness is contained at the α-terminal of the polymer chain as a polymer block.
In such a temperature-responsive polymer, a cationic monomer always exists at the α-terminal of the polymer chain, so that the degree of freedom in adjusting the position of the cationic site in the polymer chain is not high, and the cationic monomer is mainly contained in DMAEMA. Therefore, it is not always easy to adjust the positive charge intensity of the cationic site and the pH of the temperature-responsive polymer aqueous solution.
For example, when a temperature-responsive polymer is used for drug delivery (DDS), there is a possibility that the types and amounts of drugs that can be carried are limited. As a technique of DDS, for example, by applying a temperature-responsive polymer carrying a drug in a cell culture vessel and culturing cells or tissues in the cell culture vessel after application, For example, a method of gradual release of the drug can be mentioned. Here, since the temperature-responsive polymer of Patent Document 1 includes DMAEMA having a small positive charge intensity, it is not always easy to load a drug of an anionic substance, and the types and amounts of drugs that can be loaded are limited. There was a possibility.

On the other hand, in the temperature-responsive polymer of (B), NIPAM that imparts temperature responsiveness to the polymer is a neutral monomer, and the cationic monomer necessary for the formation of the cell structure (along with the anionic monomer) is NIPAM. Different monomers.
In the temperature-responsive polymer (B), it is not always necessary to have a cationic monomer at the α-terminal of the polymer chain, the position of the cationic site in the polymer chain can be freely adjusted, Since a cationic monomer can be used, it is possible to easily adjust the positive charge intensity at the cationic site and the pH of the temperature-responsive polymer aqueous solution.
According to the temperature-responsive polymer of (B), for example, when the temperature-responsive polymer is used for drug delivery (DDS), it becomes possible to increase the amount of the drug that can be carried while expanding the type of drug, As a result, the application range of a temperature-responsive polymer can be expanded.

In the temperature-responsive polymer (B), the ratio (mole) of the NIPAM unit to the total of the NIPAM unit, the cationic monomer unit, and the anionic monomer unit is preferably 0.6 to 0.9. It is more preferable that it is 7-0.9, and it is especially preferable that it is 0.9.
When other monomers are also used, the ratio (mole) of the other monomer units to the total of NIPAM units, cationic monomer units, anionic monomer units is preferably 0.001 to 0.2, More preferably, it is 0.01-0.1.

  As the temperature-responsive polymer (B), the polymer block α-terminal polymer block (for example, NIPAM homopolymer block) preferably has a number average molecular weight of 5000 Da or more, and more preferably 20000 Da or more.

The temperature-responsive polymer (B) is preferably a molecule having a number average molecular weight (Mn) of 10 to 500 kDa. Further, a molecule having a ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 1.1 to 10.0.
The molecular weight of the temperature-responsive polymer can be appropriately adjusted depending on the polymerization conditions.

  According to the temperature-responsive polymer (B), the cloud point can be lowered to, for example, room temperature (25 ° C.) or lower.

  In the above-described temperature-responsive polymer, the time until the insolubilized product of the temperature-responsive polymer formed at a temperature equal to or higher than the cloud point is redissolved under room temperature (about 25 ° C.) conditions is significantly delayed. It is presumed that this is because the obtained temperature-responsive polymer has high self-aggregation properties because of the presence of a cationic functional group and an anionic functional group in the molecule.

  In particular, the temperature-responsive polymer (B) described above has a high molecular weight NIPAM homopolymer block at the polymer chain α-terminal, and therefore, the temperature-dependent globule transition of the side chain of NIPAM is likely to occur, and the cloud point It is considered possible to effectively reduce.

  In addition, using this temperature-responsive polymer, as described later, it is possible to prepare a cell culture vessel in which the culture surface is coated with this temperature-responsive polymer.

  Furthermore, according to the temperature-responsive polymer of (B), as described later, a cell structure having a massive (pellet-like) structure can be formed by culturing cells under appropriate culture conditions.

  The ratio (C / A ratio) between the number of functional groups of the cationic functional group and the number of functional groups of the anionic functional group of the temperature-responsive polymer (B) is preferably 0.5 to 32. More preferably, it is ~ 16.

  When the C / A ratio is in the above range, the above effect of reducing the cloud point can be easily obtained. In the temperature-responsive polymer having the C / A ratio, the cationic functional group and the anionic functional group act on the intermolecular and / or intramolecular aggregation in an ionic bond in the temperature-responsive polymer, This is presumed to be a result of increasing the cohesive force of the temperature-responsive polymer.

  In addition, when the C / A ratio is in the above range, the balance between the positive charge and the negative charge in the temperature-responsive polymer can be particularly suitable, and the cytotoxicity due to the positive charge can be suppressed. It is presumed that the balance between hydrophilicity and hydrophobicity of the temperature-responsive polymer can be made particularly suitable to facilitate cell migration and orientation.

  Hereinafter, the temperature-responsive polymer (C) and the production method thereof will be described.

(Method for producing temperature-responsive polymer composition)
In the method for producing a temperature-responsive polymer composition (C), first, a mixed-type temperature-responsive polymer composition is prepared (mixture preparation step). Specifically, (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a polymer thereof and (2) 2-amino-2-hydroxymethyl-1,3-propanediol ( Tris), and (3) nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharide, curdlan and polyalginic acid, and alkali metal salts thereof. One or more anionic substances selected from the group are mixed ((2) Tris is optionally included).

  The polymer of (1) DMAEMA and / or its derivative is a temperature-responsive polymer, and its cloud point is 32 ° C. The tris in (2) serves to reduce the cloud point slightly, and / or reduce the rate at which a polymer formed at a higher temperature than the cloud point re-dissolves when cooled below the cloud point, and In addition, it is presumed to play a role of stimulating cells by positive charges derived from amino groups while maintaining hydrophilicity even in the hydrophobic polymer layer. The anionic substance (3) is presumed to play a role of enabling migration and orientation of cells to be cultured and a role of suppressing cytotoxicity.

According to this mixed temperature-responsive polymer composition, the cloud point can be reduced to room temperature (25 ° C.) or lower.
In the composition, it is presumed that the side chain of the polymer of DMAEMA and / or its derivative and Tris interact with each other (for example, a cross-linking action), and the polymer is likely to aggregate.

  Here, with respect to the above (1), as the polymer of DMAEMA and / or a derivative thereof, a molecule having a number average molecular weight (Mn) of 10 kDa to 500 kDa is preferable. Moreover, the ratio (Mw / Mn) of the weight average molecular weight (Mw) and the number average molecular weight (Mn) is preferably 1.1 to 6.0.

  In addition, as the DMAEMA derivative (1), for example, a derivative in which the hydrogen atom of the methyl group of the methacrylate is substituted with a halogen, a derivative in which the methyl group of the methacrylate is substituted with a lower alkyl group, and a hydrogen atom of the methyl group in the dimethylamino group Examples include halogen-substituted derivatives and derivatives in which the methyl group of the dimethylamino group is substituted with a lower alkyl group.

  Regarding the above (2), it is preferable that Tris is a pure substance having a purity of 99.9% or more, or that the Tris aqueous solution is made neutral or basic at the time of use by adding an alkaline substance or the like. Tris is commercially available in the form of a hydrochloride. When this is used, the pH of the aqueous Tris solution is lowered, so that the cloud point of the composition rises to about 70 ° C. Therefore, tris hydrochloride is not preferred.

  Among the anionic substances listed in (3) above, examples of the nucleic acid include DNA, RNA, and other artificial nucleic acids such as single-stranded, double-stranded, oligo, and hairpin.

The anionic substances listed in (3) above preferably have a certain size, for example, a molecular weight (M) of 1 kDa to 5,000 kDa.
When the molecular weight is within the above range, the anionic substance can ionically bond with the cationic substance and can capture the cationic substance for a long time, thereby forming stable ionic complex fine particles. . Moreover, the cytotoxicity which a cationic substance generally has by the electrostatic interaction with respect to the cell membrane surface of a cell can also be relieved.

  In addition to the anionic substances listed in (3), for example, by dehydrating and condensing a dicarboxylic acid such as oxalic acid to the 4-position amino group of poly (4-aminostyrene) which is a cationic polymer. In addition, a polymer derivative having an anionic functional group introduced and substantially functioning as an anionic substance can also be used.

  Two or more anionic substances listed in (3) above may be included.

Here, (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or its derivative polymer (2) 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) It is preferable to use a mixed temperature-responsive polymer composition having a ratio ((2) / (1)) of 1.0 or less.
The ratio ((2) / (1)) is assumed to be a weight ratio.

When the mixed temperature-responsive polymer composition in the above ratio is used, a cell structure can be easily formed in the culture step described later.
According to this composition, the balance between the hydrophilicity and the hydrophobicity of the composition can be further improved. And it is estimated that this suitable balance has adjusted the adhesiveness of the cell to a culture surface suitably, and has activated the migration and orientation of a cell.

The ratio ((2) / (1)) is preferably 0.1 or more.
By setting the ratio to 0.1 or more, the above effect of reducing the cloud point can be easily obtained. In addition, the above effect of facilitating formation of a cell structure is easily obtained.

  For the same reason as described above, the ratio ((2) / (1)) is more preferably 0.1 to 0.5.

Here, the C / A ratio (positive charge / negative charge) in the mixed temperature-responsive polymer composition is preferably 0.5 to 16.
Note that in this specification, the C / A ratio refers to the ratio of the positive charge of a substance contained in the composition to the negative charge of the substance contained in the composition. Specifically, the C / A ratio is as follows: (1) The number of moles of the polymer of DMAEMA and / or its derivative is N1, and (3) The number of moles of the anionic substance is N3. The positive charge per molecule) × N1} / {(negative charge per molecule of anionic substance) × N3}.
In this specification, when the anionic substance is DNA, the number of negative charges per molecule of the anionic substance is calculated by the number of DNA base pairs (bp number) × 2, and the molecular weight (Da) is , Bp number × 660 (average molecular weight of AT pair and CG pair).

By setting the C / A ratio to 0.5 to 16, the above effect of facilitating the formation of a tubular cell structure can be easily obtained.
It is presumed that the cytotoxicity due to the positive charge can be suppressed by suitably balancing the positive charge and the negative charge in the composition. In addition, it is presumed that the balance between hydrophilicity and hydrophobicity of the composition can be further improved to facilitate cell migration and orientation.

  For the same reason as described above, the C / A ratio is more preferably 2 to 10, and the C / A ratio is most preferably around 8.

  The temperature-responsive polymer or temperature-responsive polymer composition is preferably removed from moisture before being dissolved in an organic solvent by heat drying, freeze drying, vacuum distillation or the like.

Examples of the organic solvent that dissolves the temperature-responsive polymer or the temperature-responsive polymer composition include methanol, ethanol, n-propyl alcohol, isopropyl alcohol, 1-butanol, isobutyl alcohol, 2-butanol, t-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl-2-butanol, 2,2 -Dimethyl-1-propanol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, 3-methyl- 2-butanol, 2,2-dimethyl-1-propanol, 1-hexanol, 2-methyl Alcohols such as 2-pentanol, allyl alcohol, benzyl alcohol, salicyl alcohol; acetone, ethyl methyl ketone, diethyl ketone, methyl propyl ketone, methyl isobutyl ketone, methyl vinyl ketone, cyclohexanone, 2-methylcyclopentanone, acetophenone, Ketones such as benzophenone and isophorone; methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate, tert-butyl acetate, vinyl acetate, methyl formate, ethyl formate, propyl formate, above Examples include esters of alcohol and phosphoric acid, esters of alcohol and carbonic acid, chloroform, benzene, toluene, diethyl ether, dichloromethane, and the like.
Above all, since the surface tension is low and the contact angle to water is high, the culture surface is easily wetted, so that the culture surface can be more uniformly coated with the temperature-responsive polymer, and the solubility of the temperature-responsive polymer is even better. In view of the above, alcohols such as methanol, ethanol, n-propyl alcohol, isopropyl alcohol, 2-butanol, t-butyl alcohol, and allyl alcohol; ketones such as acetone, ethyl methyl ketone, diethyl ketone, and methyl vinyl ketone; methyl acetate, Preferred are esters such as ethyl acetate, isopropyl acetate, tert-butyl acetate, vinyl acetate; chloroform; benzene; toluene; diethyl ether; In addition, at least one selected from the group consisting of alcohols, ketones and esters is more preferable and can be dried in a short time from the viewpoint that it can be applied to polystyrene cell culture vessels and is more excellent in solubility of the temperature-responsive polymer. From the viewpoint that it can be more uniformly applied to the culture surface, for example, an organic solvent having a low boiling point (for example, a lower alcohol having 1 to 4 carbon atoms, a lower ketone having 3 to 5 carbon atoms, and an alkyl having 1 to 4 carbon atoms). At least one selected from the group consisting of alkyl acetates having a group, in particular, a lower alcohol having 1 to 4 carbon atoms, a lower alcohol having 3 to 5 carbon atoms, and an alkyl having 1 to 4 carbon atoms having a boiling point lower than that of water At least one selected from the group consisting of alkyl acetates having a group), and methanol from the viewpoint of cost and operability. Ethanol is particularly preferred. The said organic solvent may be used individually by 1 type, and may use 2 or more types together.
Since the organic solvent is excellent in solubility of the temperature-responsive polymer, the temperature-responsive polymer is not insolubilized and precipitated even when the polymer solution is at a temperature higher than the cloud point (for example, room temperature or 37 ° C.) Is dissolved. Therefore, when applying the temperature-responsive polymer, the labor for controlling the temperature of the polymer solution can be saved, and the cell culture device can be easily manufactured.

  In the above-mentioned temperature-responsive polymer solution, when the cell structure after culturing is detached from the surface of the incubator in a self-aggregating manner, a part or all of the cell structure remains without being adhered to the culture surface. From the viewpoint of facilitating exfoliation of the cell structure mass, it is preferable that a hydrophilic molecule is contained. Examples of the hydrophilic molecule include nonionic and hydrophilic molecules that do not affect the C / A ratio of the temperature-responsive polymer, such as polyethylene glycol (PEG), dimethylacrylamide (DMAA), glycerin, Triton X, and polypropylene glycol. Etc.

  The content of the temperature-responsive polymer in the temperature-responsive polymer solution is 0.00075 with respect to the polymer solution (100% by weight) from the viewpoint that the temperature-responsive polymer is likely to be uniformly coated on the entire culture surface. It is preferable that it is -0.015 weight%, and it is more preferable that it is 0.001-0.01 weight%.

  The content of the hydrophilic molecule in the temperature-responsive polymer solution is 0.00001 to 0.00015 weight with respect to the temperature-responsive polymer (100% by weight) from the viewpoint that the cell peeling phenomenon is uniform and stable. % Is preferable, and 0.00003 to 0.0001% by weight is more preferable.

When using an organic solvent that is not miscible with water, it is preferable that the temperature-responsive polymer solution does not contain water from the viewpoint that the temperature-responsive polymer is easily uniformly coated on the culture surface. The weight ratio of water in the responsive polymer solution (100% by weight) is more preferably 0.5% by weight or less, and further preferably 0.1% by weight or less. When using an organic solvent miscible with water, the weight ratio of water to the temperature-responsive polymer (100% by weight) is 20% by weight or less from the viewpoint that the temperature-responsive polymer solution is easily dried. Is more preferable, and more preferably 1% by weight or less.
The weight ratio of water can be measured by methods well known to those skilled in the art, such as gas chromatography and Karl Fischer method.

(Coating and drying process)
The manufacturing method of the cell culture device of the present embodiment includes a coating / drying step of applying the temperature-responsive polymer solution to the culture surface of the cell culture device and drying after the dissolution step.

  After the temperature-responsive polymer solution is applied to the culture surface, the polymer solution may be cast by tilting the culture surface of the cell culture vessel or extending the polymer using a spatula.

  Examples of the cell culture vessel include commercially available cell culture plates, dishes, and flasks. Examples of the material for the cell culture vessel include polystyrene, polyethylene terephthalate (PET), polypropylene, polybutene, polyethylene, and glass.

As a condition for drying the applied temperature-responsive polymer solution, from the viewpoint of more uniformly coating the culture surface with the temperature-responsive polymer, the temperature is 10 to 70 ° C. and the time is 1 to 3000 minutes under atmospheric pressure. preferable. By rapidly drying the applied temperature-responsive polymer solution, the temperature-responsive polymer can be uniformly coated on the culture surface without being biased.
The applied temperature-responsive polymer solution may be dried, for example, by allowing the cell culture vessel to stand in a 37 ° C. incubator.

  According to the manufacturing method of the cell culture device of the present embodiment, it is not necessary to perform a special treatment for coating the surface of the cell culture device with the temperature-responsive polymer, for example, radiation graft polymerization. That is, the manufacture of the cell culture device of the present embodiment can be performed at low cost by a researcher using the culture device by a simple method (without requiring a special apparatus). In addition, it is possible to easily produce a cell culture vessel in which the temperature-responsive polymer solution is uniformly coated with the dried temperature-responsive polymer without the effort of managing the temperature-responsive polymer solution or the preparation before use. .

(Cell culture device)
The cell culture device of this embodiment is manufactured by the manufacturing method of the cell culture device of this embodiment described above.

According to this cell culture vessel, cells are cultured in a general 37 ° C. incubator and matured, and a cell mass can be formed by self-aggregation with only the binding force between cells. And this cell mass can be collect | recovered, without performing cell peeling operation, such as a trypsin process.
Therefore, if cells are cultured using this cell culture device, the cell culture operation can be performed under conditions of about room temperature.

Furthermore, according to the cell culture device of an example of the present invention, a cell structure having a tubular (tubular) or pellet (lumpy) structure can be easily formed by culturing cells under appropriate culture conditions. Can be made.
This is presumed that the hydrophobic group and the cationic group that the temperature-responsive polymer has in the side chain are stimulating the cells while performing some interaction. In addition, a temperature-responsive polymer having a cationic group and an anionic group has a favorable balance between positive charge and negative charge, and suppresses cytotoxicity (the surface of the cell membrane of mammalian cells is negatively charged). Therefore, the cationic substance often has cytotoxicity), and the balance between the hydrophilicity and hydrophobicity of the temperature-responsive polymer is suitable to enable cell migration and orientation. Presumed.
Moreover, according to this cell culture device, a cell structure having a tubular (tubular) structure can be formed by culturing cells under appropriate culture conditions.

In the cell culture device of this embodiment, the amount of the temperature-responsive polymer that the culture surface of the cell culture device has per unit area is preferably 5.0 to 50 ng / mm ² , and preferably 15 to 40 ng / mm ^2. it is more preferably mm ^2. If it is the said range, the effect of making it easy to form a cell structure will be easy to be acquired.

(Cell culture method)
Below, the cell culture method using the cell culture device manufactured by the manufacturing method of this embodiment is described.
The cell culture method includes a seeding process for seeding cells in the cell culture device and a culture process for culturing the seeded cells. Seeding conditions and culture conditions can be appropriately determined by those skilled in the art based on the cell type and the purpose of the experiment. And this cell culture method is not limited to cell types, such as vascular cells, adipose stem cells, hepatocytes, chondrocytes, fibroblasts, cardiomyocytes, kidney cells, nerve cells, smooth muscle cells, chondrocytes, etc. It can be applied to various cells.

In one example of the cell culture method, the density of seeded cells is 1,500 cells / mm ² or less (seeding by adding 1.0 mL of cell suspension to a 24-well cell culture plate having a culture surface area of 200 mm ^2. In this case, it is preferably 3.0 × 10 ⁵ pieces / mL or less. The cells to be seeded are living cells.
If it is set as the said cell density, the cell structure which has a pellet-like (lump shape) structure can be formed.

  The cell culture method of the above example can be particularly suitably applied to mesenchymal cells such as vascular endothelial cells, adipocytes, adipose stem cells, and fibroblasts. In the case of primary cultured cells, an extracellular matrix derived from the basement membrane of the in vivo environment of the cells to be used may be used, and can be appropriately selected by those skilled in the art.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to the following Example at all.
In the following tests, commercially available reagents were used without further purification unless otherwise specified.

(Production of polymer 1)
To a transparent vial made of soft glass having a volume of 50 mL, 10.0 g of 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and 5 mL of water were added, and the mixture was stirred using a magnetic stirrer. The mixture (liquid) was deoxygenated by purging nitrogen gas of G1 grade high purity (purity: 99.99995%) for 10 minutes (flow rate: 2.0 L / min). The DMAEMA used contained 0.5% by weight of methylhydroquinone (MEHQ) which is a polymerization inhibitor.
Thereafter, the reaction product was polymerized by irradiating the reaction product with ultraviolet rays for 22 hours using a round black fluorescent lamp (manufactured by NEC, model number: FCL20BL, 18W). The reaction product became viscous after 5 hours and solidified after 15 hours to obtain a polymer as a reaction product. The reaction product was dissolved in 2-propanol and the solution was transferred to a dialysis tube. Then, dialysis was performed for 72 hours to purify the reaction product.
The solution containing the reaction product is filtered through a 0.2 μm filter made of cellulose mixed ester (manufactured by Toyo Roshi Kaisha, model number: 25AS020), and the resulting filtrate is freeze-dried to obtain a temperature-responsive (homo) polymer. Obtained (yield: 6.8 g, conversion: 68%). The number average molecular weight (Mn) of this polymer was measured using GPC (manufactured by Shimadzu Corporation, model number: LC-10vp series) with polyethylene glycol (manufactured by Shodex, TSK series) as a standard substance, and Mn = 160,000. (Mw / Mn = 3.0) was determined (Polymer 1).

The nuclear magnetic resonance spectrum (NMR) of polymer 1 was measured using a nuclear magnetic resonance apparatus (manufactured by Varian, model number: Gemini 300) using heavy water (D ₂ O) as a standard substance. The following shows typical peaks common to polymer 1.
¹ H-NMR (in D ₂ O) δ 0.8-1.2 (br, -CH ₂ -C (CH ₃ )-), 1.6-2.0 (br, -CH ₂ -C (CH ₃ )-), 2.2-2.4 (br, -N (CH ₃ ) ₂ ), 2.5-2.7 (br, -CH ₂ -N (CH ₃ ) ₂ ), 4.0-4.2 (br, -O-CH _2- ).
Here, the number of protons of the methyl group (δ 0.8-1.2) in the main chain (three per monomer molecule in the case of DMAEMA homopolymer) A and the methyl protons of the dimethylamino group (δ 2.2-2.4) in the side chain The number of amino groups in the side chain and the side chain ester bond hydrolysis proceeding simultaneously with the polymerization reaction, from the number (6 in the case of DMAEMA homopolymer, 6 per monomer molecule) B The ratio with the number of functional groups of the carboxyl group in the side chain was calculated.
As a result, in the case of polymer 1, it was 94: 6. When this is converted into the C / A ratio referred to as an ionic complex in a two-component mixed system containing a cationic polymer and an anionic polymer, the C / A ratio is 15.6.

The cloud point of polymer 1 was measured by the following method.
A 3% aqueous solution of polymer 1 was prepared, and the absorbance at 660 nm of this aqueous solution was measured between 20 ° C and 40 ° C.
As a result, the aqueous solution was transparent at 20 ° C. to 30 ° C. and the absorbance was almost 0. However, white turbidity was observed in the aqueous solution from around 31 ° C., and the absorbance rapidly increased at 32 ° C. This confirmed that Polymer 1 had a cloud point of about 32 ° C.
When the temperature of polymer 1 was raised to 37 ° C., the aqueous polymer solution was suspended with good responsiveness, and then the entire aqueous solution was solidified. When this solidified product was maintained at room temperature (25 ° C.), it remained solidified for several tens of hours. Thereafter, the solidified product was gradually dissolved and changed into a homogeneous aqueous solution. When the solidified polymer was cooled to 4 ° C., it quickly dissolved. And even if it repeated the said temperature raising and temperature-decreasing operation, the change in responsiveness did not occur, and it was confirmed that the polymer reversibly causes a phase transition.

Example 1
Polymer 1 was dissolved in methanol to prepare a temperature-responsive polymer solution (final concentration 15 μg / mL). 200 μL of this solution was added to each well of a polystyrene 24-well cell culture plate (manufactured by Iwaki Co., Ltd., microplate, model number: 3815-024, bottom area per well: 200 mm ² ) at room temperature, A 24-well cell culture in which a culture bench is covered with the dried polymer 1 by leaving it in the vicinity of the suction hole in a clean bench (Sanyo Electric Co., Ltd., MHE-131AJ) and drying it for 24 hours to completely evaporate methanol. A plate (cell incubator) was obtained.

(Example 2)
Except that polymer 1 and hydrophilic molecules (polyethylene glycol, PEG300, manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in methanol (final concentrations were 15 μg / mL and 0.75 μg / mL, respectively), the same as in Example 1 As a result, a 24-well cell culture plate (cell culture vessel) obtained by coating the culture surface with the dried polymer 1 was obtained.

(Comparative Example 1)
Polymer 1 was dissolved in water to prepare a temperature-responsive polymer aqueous solution (final concentration 15 μg / mL). The temperature of this aqueous solution is controlled so as not to exceed the cloud point, and is added to each well of a polystyrene 24-well cell culture plate (manufactured by Iwaki Co., Ltd., microplate, model number: 3815-024, bottom area per well: 200 mm ² ). 200 μL each was added at room temperature, and the 24-well cell culture plate was tilted and quickly cast on the entire culture surface. Thereafter, a temperature-responsive polymer aqueous solution is dried for 96 hours in a 37 ° C. incubator (manufactured by Sanyo Electric Co., Ltd., MCO-5AC), and the culture surface is coated with the dried polymer 1 (24-well cell culture plate (cell culture) Was obtained.

[Evaluation]
(Handability in the manufacture and use of cell culture vessels)
The handling property in the manufacture and use of the cell culture device was evaluated according to the following criteria.
○ (good): The solvent of the solution containing the temperature-responsive polymer volatilized in a short time.
X (poor): It took time to volatilize the solvent of the solution containing the temperature-responsive polymer.

(Uniformity of polymer coating on the culture surface)
Each well of the 24-well cell culture plate obtained in Examples and Comparative Examples is observed with a scanning electron microscope (trade name “S4300”, manufactured by Hitachi, Ltd.), and whether the temperature-responsive polymer is uniformly coated, Was observed. Then, the uniformity of the polymer coating on the culture surface was evaluated according to the following criteria.
○ (Good): The polymer was uniformly coated on the culture surface.
Δ (ordinary): The polymer was agglomerated and coated on the outer peripheral edge of the culture surface, and the center was hardly coated.
X (Poor): The polymer was not coated on the culture surface.

(Peelability of cell structure)
In each well of the 24-well cell culture plate obtained in Examples and Comparative Examples, rat subcutaneous fat-derived mesenchymal stem cells were placed in a complete medium (Dulbecco's modified Eagle medium (DMEM) + 10% fetal calf serum) at room temperature. (FCS) solution, DMEM: manufactured by Gibco, Model No. 11965, FCS: manufactured by BI, lot number 715929), and cell suspension adjusted to a cell density of 3.0 × 10 ⁵ cells / mL, 1 mL each Added (1,500 pieces / mm ² ).
The cells were cultured for 24 hours in a cell culture incubator at 37 ° C. Three hours after seeding, the cells adhered to the entire culture surface (100% confluent). After 10 hours of culturing, all the cells are once detached from the outer periphery of each well, and the cells are agglomerated in one place by the operation of shrinking while curling the sheet. Aggregates to form a lump, and then the cell structure peels from the culture surface. And the peelability of the cell structure was evaluated according to the following criteria.
A (excellent): no cells remained on the culture surface, and the cell structure was detached as a single block (see FIG. 3).
○ (Good): Cells adhered to a small part of the culture surface without peeling (see FIG. 4. In FIG. 4, the tadpole-shaped tail part adhered to the cells without peeling. is there.).
Δ (Normal): Cells were detached at the outer peripheral edge of the culture surface, but cells in the central part were hardly detached (see FIG. 5. The part surrounded by is the part where the cells are not detached).
X (Poor): The cell structure was not detached at all.

Claims (7)

A dissolution step of preparing a temperature-responsive polymer solution by dissolving a temperature-responsive polymer or a temperature-responsive polymer composition in an organic solvent;
Applying and drying the temperature-responsive polymer solution on the culture surface of the cell incubator, and drying,
A method for producing a cell culture device, comprising:   The method for producing a cell culture device according to claim 1, wherein the organic solvent is at least one selected from the group consisting of alcohol, ketone and ester.   The organic solvent is at least one selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, lower ketones having 3 to 5 carbon atoms, and alkyl acetates having an alkyl group having 1 to 4 carbon atoms, The manufacturing method of the cell culture device of Claim 2.   The method for producing a cell culture device according to any one of claims 1 to 3, wherein the temperature-responsive polymer solution further contains a hydrophilic molecule. The temperature-responsive polymer is a repeating unit (A) represented by the formula (I)
And the repeating unit (B) represented by the formula (II)
The manufacturing method of the cell culture device of any one of Claims 1-4 which is a temperature-responsive polymer containing this. The temperature-responsive polymer comprises a homopolymer region of the repeating unit (A);
The manufacturing method of the cell culture device of Claim 5 which is a polymer which has a copolymer area | region of the said repeating unit (A) and the said repeating unit (B).   A cell culture device manufactured by the method for manufacturing a cell culture device according to any one of claims 1 to 6.