JP2021045117A - Cell culture member and production method thereof - Google Patents

Abstract

To provide cell culture members that have low solubility in water and low cytotoxicity, form spheroids of appropriate size, prevent inner cell necrosis, are easy and cheap to make, and in which spheroids are not removed during medium replacement, and to provide production methods thereof.SOLUTION: Provided is a cell culture member having a base layer and a surface layer for culturing adherent cell spheroids having a diameter of 50 to 250 μm and having no differentiation function, the surface layer containing a block polymer (A) in which an acrylic polymer moiety (A1), which is formed from monomers with an average value of water/1-octanol partition coefficient of 0 or more and 2 or less, and a polyethylene oxide moiety (A2) are connected via any of the structures of the following equations (IA) to (IC), the polymer (A) having the mass of the polymer (A)/[the total mass of (A1) and (A2)] of 0.01% or more and less than 1%, and the base layer containing any of polyolefin, polycycloolefin, polystyrene, glass, and silicone.SELECTED DRAWING: Figure 1

Description

The present invention relates to a novel cell culture member and a method for producing the same.

Collagen, polystyrene and polymethylmethacrylate are generally known to be good in the development of cell culture components, which are one of the tools for drug discovery research and regenerative medicine research, and polystyrene is particularly cytotoxic. It has advantages in low efficiency, economy, and processability, and in the current tissue culture, polystyrene treated with hydrophilic treatment is used.

However, the cells that form each organ such as the liver, pancreas, skin, and blood vessels exist in the living body as cell aggregates in which the cells form a three-dimensional network with each other, and a general resin such as polystyrene is used. Although cell elongation and proliferation are observed in the culture member made of the above, cell aggregates (spheroids) are not formed.

Against the background of these problems, Patent Document 1 proposes a method of culturing in a floating state in a container in which a resin layer having low cell adhesion is formed as a method for performing three-dimensional culture in which spheroids are formed. There is. Further, a method of coating a resin having low cell adhesion into a container has been widely studied. For example, in Patent Document 2, an acrylic resin in which a polyethylene oxide portion (hereinafter, may be abbreviated as PEG) is incorporated in a side chain is used. It has been proposed, and Patent Document 3 discloses an acrylic resin in which a PEG chain is incorporated in a main chain by the present inventors.

Further, Patent Document 4 discloses a method of forming a spheroid using a base material having a nanoimprinted uneven structure, and Patent Document 5 discloses a method of forming a spheroid on a base material having a special shape such as a micro pattern. Is disclosed.

Japanese Unexamined Patent Publication No. 2008-061609 Japanese Patent No. 4746984 Japanese Patent No. 5831666 Japanese Patent No. 6113565 Japanese Unexamined Patent Publication No. 2006-67987

However, in the case of the resin having low cell adhesion as described in Patent Documents 1, 2 and 3, the cell motility becomes too high, so that the cell adhesion becomes high. This makes it difficult to control the size of the spheroids formed, and if the size of the spheroids becomes too large, the cells in the center of the spheroids may die. Therefore, there has been a problem in expressing the function of the cell in cells cultured in vitro and using it in applications such as drug discovery screening in which it is necessary to maintain it for a long period of time. In addition, when cells are cultured for a long period of time, it is necessary to replace the medium regularly, but when a resin having low cell adhesion is used for the surface layer of the substrate, spheroids are formed in a state of being suspended in the medium. Therefore, there is a problem that spheroids are removed together with the medium when the medium is removed at the time of medium exchange.

Further, the base material having a fine structure as described in Patent Documents 4 and 5 has a problem that the cost associated with the imprint processing and the plasma etching processing is high and the work is complicated.

Therefore, the problem to be solved by the present invention is a cell culture member having low solubility in water and low cytotoxicity, and capable of preventing necrosis of internal cells by forming spheroids in an appropriate size. It is an object of the present invention to provide a cell culture member which is easy and inexpensive to prepare and in which spheroids are not removed at the time of medium exchange, and a method for producing the same.

As a result of diligent studies, the present inventors avoided internal necrosis of spheroids when cell culture was performed on a cell culture member coated with a polymer having a PEG chain in the main chain of 0.01% or more and less than 1%. Moreover, it was found that the spheroid was not removed at the time of medium exchange, and the present invention was completed. Furthermore, they have found that they can provide a cell culture member capable of forming spheroids even after gamma ray sterilization, and have completed the present invention.

（２）前記基材層は、ポリオレフィン、ポリシクロオレフィン、ポリスチレン、ガラス、およびシリコーンからなる群より選ばれる少なくとも１種を含む。 That is, the present invention is a cell culture member having a base material layer and a surface layer for culturing adherent cell spheroids having a diameter of 50 to 250 μm and having no differentiation function, and the following (1) and ( A cell culture member that satisfies 2).
(1) The surface layer has an acrylic polymer portion (A1) formed from an acrylic monomer having an average value of water / 1-octanol partitioning coefficient (LogP) of 0 or more and 2 or less, and a polyethylene oxide portion (A2). ) Contains a block polymer (A) formed by bonding via any of the structures of the following formulas (IA) to (IC), and the block polymer (A) is the mass of the polyethylene oxide moiety (A2). / [Total mass of acrylic polymer moiety (A1) and polyethylene oxide moiety (A2)] is 0.01% or more and less than 1%.

(2) The base material layer contains at least one selected from the group consisting of polyolefin, polycycloolefin, polystyrene, glass, and silicone.

The present invention also relates to the cell culture member having an average surface roughness Ra of the surface layer of 10 nm or less.

式中、ｍ、ｎは１以上の整数を示す。 Further, in the present invention, the block polymer (A) has a polyethylene oxide moiety (A2) represented by the following general formula (II) and has a mass average molecular weight of 5,000 to 100,000. The present invention relates to the cell culture member, which is a block copolymer obtained by polymerizing an acrylic monomer using a system polymerization initiator.

In the formula, m and n represent integers of 1 or more.

The present invention also relates to the cell culture member, wherein the block polymer (A) has a Tg of −70 ° C. to 70 ° C. and contains C element, H element, and O element in the side chain.

The present invention also relates to the cell culture member, which is a dish.

The present invention also relates to the cell culture member for drug discovery screening.

The present invention is a method for producing a cell culture member having a base material layer and a surface layer for culturing adherent cell spheroids having a diameter of 50 to 250 μm and having no differentiation function.
The acrylic polymer moiety (A1) formed from an acrylic monomer having an average value of water / 1-octanol partitioning coefficient (LogP) of 0 or more and 2 or less and the polyethylene oxide moiety (A2) are represented by the following formula (IA). ) To the block polymer (A) bonded via any of the structures (IC), and the mass of the polyethylene oxide moiety (A2) / [acrylic polymer moiety (A1) and polyethylene oxide moiety (A2). For cell culture, the base material layer is coated with a coating agent containing 0.1 to 10% by mass of the block polymer (A) having a total mass of 0.01% or more and less than 1% to form a surface layer. Regarding the method of manufacturing a member.

The present invention also relates to a method for producing the cell culture member, which further includes a step of sterilizing with gamma rays.

The present invention also relates to a method for producing the cell culture member, wherein the method of coating the base material layer with the coating agent is a spin coating method.

According to the present invention, it is a cell culture member having low solubility in water and low cytotoxicity, and capable of preventing necrosis of internal cells by forming spheroids in an appropriate size, and is easy and inexpensive to prepare. It is possible to provide a cell culture member from which spheroids are not removed at the time of medium exchange and a method for producing the same.

FIG. 1 is an optical micrograph showing a spheroid observed on a dish prepared in an example of the present invention. Five or more spheroids are observed per field of view. FIG. 2 is an optical micrograph of cells observed on a comparative dish. No spheroids were observed around the visual field, and cell adhesion and progression were observed.

<Block polymer (A)>
The block polymer (A) is an acrylic polymer moiety (A1) formed from a monomer having an average value of water / 1-octanol partition coefficient (cLogPow: hereinafter LogP) of 0 or more and 2 or less (hereinafter, 0 to 2). ) And a polyethylene oxide moiety (A2).

LogP is one of the numerical values representing the properties of the chemical substance, and is a constant value that does not depend on the amount of addition. The concentration of each phase is shown by its common logarithm for the case where the target substance is in an equilibrium state in a system in which the aqueous phase and the octanol phase are in contact with each other in a mixed solution of water and 1-octanol. .. As LogP increases, hydrophobicity tends to increase relatively, and as LogP decreases, hydrophilicity tends to increase relatively.

The measurement of LogP can be generally carried out by the flask dipping method described in JIS Japanese Industrial Standard Z7260-107 (2000). In addition, LogP can be estimated by a computational chemistry method or an empirical method instead of the actual measurement. As for the method and software used for the calculation of LogP, known ones can be used, but in the present invention, LogP is obtained by using the program incorporated in the system of CambridgeSoft: ChemdrawPro11.0.

Since the block polymer (A) of the present invention has an acrylic polymer portion (A1) formed from a monomer having an average value of LogP of 0 or more and 2 or less (hereinafter, 0 to 2). Solubility in water is controlled and fat solubility is improved.

Further, in the block polymer (A) of the present invention, the acrylic polymer portion (A1) and the polyethylene oxide portion (A2) have a structure (bonded structure) of any of the following formulas (IA), (IB) or (IC). ). Since the polyethylene oxide moiety (A2) is incorporated in the main chain, microphase separation is induced and low antigenicity can be imparted by changing the surface aggregation structure.

The block polymer (A) has a mass of the polyethylene oxide moiety (A2) / [total mass of the acrylic polymer moiety (A1) and the polyethylene oxide moiety (A2)] = 0.01% or more and less than 1%. (A2) / [(A1) + (A2)] = 0.1% or more and 0.5% or less.
The balance between the polyethylene oxide moiety (A2), which is rich in hydrophilicity, and the acrylic polymer moiety (A1), which is rich in hydrophobicity, suppresses aggregation of suspended spheroids and prevents internal necrosis. Further, (A2) is controlled in a low range of 0.01% or more and less than 1%, and the design is such that the hydrophilicity does not become too high, so that spheroid removal at the time of medium replacement can be prevented.

The method for synthesizing the block polymer (A) having the bonding structure of any one of the formula (IA), (IB) or (IC) is not particularly limited. As a method for synthesizing a block polymer having a binding structure represented by the above formula (IA), for example, 4,4'-azobis (4-cyanovaleric acid) as a radical polymerization initiator (for example, Fujifilm Wako Pure Chemical Industries, Ltd.) When an acrylic polymer is synthesized using a commercially available product "V-501", etc.), an acrylic resin having a carboxyl group at the terminal can be obtained. By adding polyethylene glycol as a raw material constituting the PEG moiety and subjecting it to an esterification reaction, it is possible to obtain a block polymer in which the acrylic polymer moiety and the PEG moiety are bonded by the above formula (IA). it can.

Alternatively, as a radical polymerization initiator when synthesizing an acrylic polymer, a polymer azo polymerization initiator having a structural unit containing a PEG moiety (A2) and an azo group represented by the following formula (II) is preferably used. can do. In the formula, m and n are each independently an integer of 1 or more. The polymer azo polymerization initiator has a structure in which a polymer segment and an azo group (-N = N-) are repeatedly bonded, and in the present embodiment, the polymer azo polymerization initiator contains a PEG block as the polymer segment. By using the above, a block polymer can be easily synthesized.

When a high molecular weight azo polymerization initiator is used, the mass average molecular weight of the block polymer can be adjusted by appropriately changing the ratio of the total number of moles of the acrylic monomer to the number of moles of the azo group contained in the initiator. it can. For example, when the ratio of the total number of moles of the acrylic monomer to the number of moles of the azo group contained in the polymer azo polymerization initiator is 200, a 200-mer acrylic polymer is incorporated between the PEG chains. Therefore, it is possible to impart a high cohesive force due to the entanglement of the polymers.

The mass average molecular weight of the polymer azo polymerization initiator is preferably about 5,000 to 100,000, and more preferably about 10,000 to 50,000. The molecular weight of the PEG moiety of the initiator is preferably about 800 to 10,000, more preferably about 1,000 to 8,000.
Further, m is preferably 15 to 200, more preferably m is 20 to 100, preferably n is 3 to 50, and more preferably n is 4 to 30.

Since the polymer azo polymerization initiator has a PEG moiety (A2), it is soluble in water, alcohol, and organic solvents, and block polymers can be synthesized by solution polymerization, emulsion polymerization, or dispersion polymerization. It is possible. Further, since the molecular chain skeleton has a polymerization initiation portion (radical generation portion: −N = N−), it is not necessary to use a separate polymerization initiator, and further, radicals are compared with the terminal reactive macromonomer. It has the characteristics of high reactivity and stability.
The polymer azo polymerization initiator is: C (CH ₃ ) CN- (CH ₂ ) _2- COO- (CH ₂ CH ₂ O) _m- CO- (CH ₂ ) _2- C (CH ₃ ) CN. A radical as shown in the above is generated, and an acrylic monomer described later is polymerized. Then, the acrylic polymer portion (A1) formed from the acrylic monomer and the radical-derived portion are bonded to form a main chain to form a block polymer (A). The PEG moiety (A2) is derived from some of the radicals.
As a specific example of the polymer azo polymerization initiator, the polymer azo initiator VPE0201 manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. ( _{the molecular weight of the (CH 2} CH ₂ O) _m portion of the above formula (II) is about 2000, n is about 6) and the like are exemplified.

In addition to the high molecular weight azo polymerization initiator, an azo initiator such as 2,2'-azobisisobutyronitrile and an organic peroxide initiator such as benzoyl peroxide can be used in combination. By using these other initiators in combination, the initiation efficiency can be increased, the acrylic polymer portion (A1) can be efficiently incorporated into PEG, and the residual monomer can be reduced.

At the time of synthesizing the block polymer, the molecular weight and the terminal structure may be controlled by using mercaptans such as lauryl mercaptan and n-dodecyl mercaptan, and chain transfer agents such as α-methylstyrene dimer and limonene, depending on the intended use.

As a method for synthesizing a block polymer having a bonded structure represented by the formula (IB), for example, an acrylic monomer is polymerized using an azo polymerization initiator having a hydroxy group, and an acrylic polymer having a hydroxy group at the terminal. It can be produced by a method including a step of obtaining a block and a step of reacting the acrylic polymer block and polyethylene glycol with a bifunctional isocyanate compound.

For example, 2,2'-azobis [N- (2-hydroxyethyl) -2-methylpropanamide] (for example, a commercially available product "VA-086" of Fujifilm Wako Pure Chemical Industries, Ltd.) is used as a radical polymerization initiator. When an acrylic polymer is synthesized using this, an acrylic resin having a hydroxyl group at the terminal is obtained. By adding polyethylene glycol as a raw material constituting the PEG moiety to this and subjecting it to a urethanization reaction using a bifunctional isocyanate compound, it is possible to obtain a block polymer in which the acrylic polymer moiety and the PEG moiety are bonded. it can.
Examples of the bifunctional isocyanate compound used in the urethanization reaction include 2,2'-diphenylmethane diisocyanate, 2,4'-diphenylmethane diisocyanate, 4,4'-diphenylmethane diisocyanate, 2,4-tolylene diisocyanate, and 2, Examples thereof include 6-tolylene diisocyanate, hexamethylene diisocyanate, and isophorone diisocyanate.

As a method for synthesizing a block polymer having a bonded structure represented by the formula (IB), for example, an acrylic monomer is polymerized with an azo polymerization initiator having a carboxyl group to obtain an acrylic polymer block having a carboxyl group. It can be produced by a method including a step of obtaining the acrylic polymer block and a step of subjecting the acrylic polymer block to an esterification reaction of polyethylene glycol.

For example, as a radical polymerization initiator, 2,2'-azobis [N- (2-carboxyethyl) -2-methylpropionamidine] tetrahydrate (for example, a commercial product "VA-057" of Fujifilm Wako Pure Chemical Industries, Ltd. When an acrylic polymer is synthesized using (etc.), an acrylic resin having a carboxyl group at the terminal can be obtained. By adding polyethylene glycol as a raw material constituting the PEG moiety and subjecting it to an esterification reaction, a block polymer in which the acrylic polymer moiety and the PEG moiety are bonded can be obtained.

In addition, when obtaining a block polymer having a binding structure of the formula (IB) or (IC), the above-mentioned "other initiator" can also be appropriately used in combination. In addition, a chain transfer agent can also be used as appropriate.

In the present invention, the mass of the raw material containing the polyethylene oxide structure is applied as it is to the mass of the polyethylene oxide portion (A2). For example, when it has a bonded structure represented by the formula (IA), it is the mass of the polymer azo-based polymerization initiator containing the polyethylene oxide moiety (A2) and the azo group. The mass of the acrylic polymer portion (A1) is the total amount of the acrylic monomers used for the polymerization.
That is, when synthesized using 99.5 parts by mass of the monomer and 0.5 parts by mass of the polymer azo-based polymerization initiator having the polyethylene oxide portion (A2), the mass of the polyethylene oxide portion (A2) / [acrylic polymer portion The total mass of (A1) and the polyethylene oxide portion (A2)] is 0.5%.

<Monomer>
Next, the acrylic monomer which is the raw material of the acrylic polymer portion (A1) of the block polymer (A) will be described. In the present invention, the acrylic monomer means both an acrylic monomer and a methacrylic monomer.
The average value of the water / 1-octanol partition coefficient (LogP) of the acrylic monomer is 0 or more and 2 or less. When synthesizing using an acrylic monomer having an average value of LogP lower than 0, the block copolymer has high hydrophilicity and solubility in water is improved, so that a stable coating film cannot be formed.
On the other hand, when synthesizing using an acrylic monomer having an average value of LogP higher than 2, the solubility of the block copolymer in water is suppressed, but the hydrophobicity is too high, so that spheroids cannot be formed. Furthermore, LogP has a high correlation with biological activity (toxicity), and the subject of the present invention is to suppress internal necrosis due to aggregation of suspended spheroids and maximize the effect of biological safety. I can't.

In the present invention, the average value of LogP is a value obtained by averaging the LogP of each monomer used by the mass% of each monomer. That is, when a monomer having 0 LogP and a monomer having 2 LogP are charged at a ratio of 50:50 mass%, the average value of LogP is 1.

In the present invention, as long as the LogP of the acrylic monomer used is in the range of 0 to 2, it can be used as a raw material for the homopolymer or copolymer moiety. Further, even if the LogP of the acrylic monomer used is outside the range of 0 to 2, if the average value of LogP including other acrylic monomers is in the range of 0 to 2, it is used as a raw material for the copolymer portion. be able to.

Examples of the monomer having a water / 1-octanol partition coefficient (LogP) of 0 or more and 2 or less include, for example.
Alkyl acrylates with 1 to 4 carbon atoms in the alkyl group;
Alkyl methacrylate having 1-3 carbon atoms in the alkyl group;
Methoxymethyl (meth) acrylate, 2-methoxyethyl (meth) acrylate, ethoxymethyl (meth) acrylate, ethoxyethyl (meth) acrylate, propoxymethyl (meth) acrylate, propoxyethyl (meth) acrylate, 2- (2-methoxy) Ethoxy) ethyl (meth) acrylate, 2- [2- (2-methoxyethoxy) ethoxy] ethyl (meth) acrylate, 2- (2-ethoxyethoxy) ethyl (meth) acrylate, 2- [2- (2-ethoxy) ethoxy Ethoxy) ethoxy] ethyl (meth) acrylate, tetrahydrofurfuryl (meth) acrylate and other alkoxy group-containing monomers;
(Meta) Vinyl group-containing monomers such as acrylonitrile, vinyl acetate, butadiene, and isoprene;
Amide group-containing monomers such as N-vinylpyrrolidone, N-vinyl-ε-caprolactam, 1-vinylimidazole, N-isopropylacrylamide, N, N-dimethylacrylamide;
And so on.
These may be used alone or in combination of two or more. Of these compounds, 2-methoxyethyl acrylate and tetrahydrofurfuryl acrylate are particularly preferable from the viewpoint of economy and operability.

The block polymer (A) is preferably copolymerized with a monomer having a functional group capable of reacting with a cross-linking agent. By forming the crosslinked coating film, peeling and elution of the block polymer (A) can be suppressed.
As the monomer having a LogP of 0 to 2 and having a functional group, a carboxyl group-containing monomer, a hydroxyl group-containing monomer, an epoxy group-containing monomer, an amino group-containing monomer, an isocyanate group-containing monomer and the like can be used.
For example, a copolymer having a carboxyl group introduced therein can be crosslinked with an epoxy compound, an aziridine compound, a carbodiimide compound, a metal chelate compound, or an N-hydroxyethylacrylamide compound. The copolymer into which a hydroxyl group has been introduced can be crosslinked with an isocyanate compound, a carbodiimide compound, or the like. The copolymer into which the amino group has been introduced can be crosslinked with an epoxy compound. The copolymer into which an isocyanate group has been introduced can be crosslinked with a hydroxyl group-containing compound. The amount of these monomers having a functional group serving as a cross-linking point is preferably 10% by mass or less based on 100% by mass of the total of all the monomers. By using it in an amount of 10% by mass or less, a coating film having an appropriate crosslinking density can be obtained when a crosslinking agent is used in combination.

The acrylic polymer moiety (A1) is a homopolymer moiety or a copolymer moiety formed only from monomers having a water / 1-octanol partition coefficient (LogP) of 0 to 2. Further, a block copolymer may be obtained by using only one kind of monomer having LogP 0 to 2, or a block copolymer may be obtained by polymerizing using a plurality of kinds of monomers having LogP 0 to 2. May be good.

Among the monomers having LogP in the range of 0 to 2, examples of the monomer having no functional group serving as a cross-linking point include, for example, an acrylate having an alkyl group having 5 to 20 carbon atoms and an alkyl group having 4 to 20 carbon atoms. Examples thereof include 20 methacrylates and vinyl group-containing monomers such as styrene.

Among the monomers having LogP in the range of 0 to 2, examples of the monomer having a functional group serving as a cross-linking point include, for example, a carboxyl group-containing monomer such as maleic acid, 4-hydroxystyrene, and N-hydroxyethyl (meth) acrylamide. Examples thereof include hydroxyl group-containing monomers.

Further, in the present invention, a polyfunctional monomer may be copolymerized in order to introduce a crosslinked structure into the block polymer (A).
When cross-linking while copolymerizing, the cross-linking ratio increases as the amount of polyfunctional monomer used increases, and the possibility of gelation during the reaction also increases. Therefore, the amount of the polyfunctional monomer is preferably in the range of 0.01 to 10% by mass, more preferably 0.1 to 5% by mass, based on 100% by mass of all the monomers. When the block polymer (A) is crosslinked with a polyfunctional monomer, it becomes poorly water-soluble, so that peeling and elution from the substrate can be reduced as in the case of using a crosslinking agent.

In the present invention, the block polymer (A) can be synthesized by a known method. For example, solution polymerization, bulk polymerization, emulsion polymerization, dispersion (precipitation) polymerization and the like are preferable, and solution polymerization and dispersion (precipitation) polymerization are more preferable.

The mass average molecular weight of the block polymer (A) is preferably 3,000 to 1,000,000, more preferably 5,000 to 500,000 from the viewpoint of handleability and coating on a substrate. is there.

The glass transition temperature of the block polymer (A) is preferably −70 ° C. to 70 ° C., more preferably −50 ° C. to 50 ° C. By adjusting the glass transition temperature of the block polymer (A) in the range of −70 ° C. to 70 ° C., good coating property to the cell culture member is possible, and adhesion to the cell culture member can be imparted. it can.
The glass transition temperature can be adjusted by appropriately adjusting the monomers constituting the acrylic polymer portion (A1). For example, by increasing the ratio of polyalkoxyalkyl (meth) acrylate such as 2-methoxyethyl (meth) acrylate, the flexibility peculiar to the side chain ether bond can be imparted, so that the glass transition temperature is −50 ° C. It can be prepared in a range close to.

As described above, the block polymer (A) of the present invention can optimize the molecular weight, the type of side chain functional group, and the amount of side chain functional group introduced. Among them, the block polymer (A) having a Tg of −70 ° C. to 70 ° C. and containing an element C, an element H, and an element O in the side chain is preferable.

<Crosslinking agent>
Next, the cross-linking agent that can be used in the present invention will be described. Examples of the cross-linking agent that can be used in the present invention include those having at least one functional group selected from an epoxy group, an isocyanate group, and an aziridinyl group, a metal chelate compound, a carbodiimide group-containing compound, and the like. These cross-linking agents can be used for the purpose of increasing the elastic modulus and resistance of the coating film, and for adjusting the adhesive force.

[Crosslinking agent with epoxy group]
The cross-linking agent having an epoxy group used in the present invention is not particularly limited as long as it has two or more epoxy groups in one molecule.
Examples of the cross-linking agent having a bifunctional epoxy group include ethylene glycol diglycidyl ether, polyethylene oxide diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, tetramethylene glycol diglycidyl ether, and polytetramethylene glycol diglycidyl. Aliper epoxy compounds such as ether, 1,6-hexanediol diglycidyl ether, neopentyl glycol diglycidyl ether, polybutadiene diglycidyl ether, bisphenol A type epoxy resin, bisphenol F type epoxy resin, bisphenol S type epoxy resin, biphenol type Aromatic epoxy compounds such as epoxy resins, dihydroxybenzophenone diglycidyl ethers, resorcinol diglycidyl ethers, hydroquinone diglycidyl ethers, dihydroxyanthracene-type epoxy resins, bisphenol full orange glycidyl ethers, N, N-diglycidyl aniline, and the aromatics described above. Examples thereof include hydrogenated epoxy compounds and alicyclic epoxy compounds such as hexahydrophthalic acid diglycidyl ester.
Examples of the cross-linking agent having three or more epoxy groups include triglycidyl isocyanurate, trisphenol type epoxy compound, tetrakisphenol type epoxy compound, phenol novolac type epoxy compound and the like.

[Crosslinking agent with isocyanate group]
The cross-linking agent having an isocyanate group used in the present invention is not particularly limited as long as it is a compound having two or more isocyanate groups in one molecule.
Examples of the bifunctional isocyanate compound include 2,2'-diphenylmethane diisocyanate, 2,4'-diphenylmethane diisocyanate, 4,4'-diphenylmethane diisocyanate, 2,4-tolylene diisocyanate, 2,6-tolylene diisocyanate, and hexa. Examples thereof include methylene diisocyanate and isophorone diisocyanate.
Examples of the trifunctional isocyanate compound include the trimethylolpropane adduct of diisocyanate described above, the Biuret body reacted with water, and the trimer having an isocyanurate ring.
Further, the isocyanate group in the cross-linking agent having an isocyanate group may or may not be blocked.
The blocked isocyanate cross-linking agent used in the present invention may be a blocked isocyanate compound in which the isocyanate group in the isocyanate compound is blocked with ε-caprolactam, MEK oxime, cyclohexanone oxime, pyrazole, phenol or the like, and is particularly limited. It is not something that is done.

[Crosslinking agent with aziridinyl group]
The aziridine compound used in the present invention is not particularly limited as long as it is a compound having two or more aziridinyl groups in one molecule. Examples of the aziridine compound include 2,2'-bishydroxymethylbutanoltris [3- (1-aziridinyl) propionate], 4,4-bis (ethyleneiminocarbonylamino) diphenylmethane and the like.

[Metal chelate compound]
Examples of the metal chelate compound used in the present invention include organic aluminum compounds such as aluminum chelate compounds, aluminum alkoxide compounds and aluminum acylate compounds, organic titanium compounds such as titanium chelate compounds, titanium alkoxide compounds and titanium acylate compounds, and zirconium. Examples thereof include organic zirconium compounds such as chelate compounds, zirconium alkoxide compounds and zirconium acylate compounds.

[Carbodiimide group-containing compound]
As the carbodiimide group-containing compound used in the present invention, the carbodilite series of Nisshinbo Holdings Co., Ltd. can be used, and aqueous types such as V-02, V-04, and V-06, V-01, and V-03 can be used. , V-05, V-07, V-09 and other oil-based types.

[Β-Hydroxyalkylamide group-containing compound]
The β-hydroxyalkylamide group-containing compound is not particularly limited as long as it is a compound containing a β-hydroxyalkylamide group in the molecule. Examples of the β-hydroxyalkylamide group-containing compound include various compounds such as N, N, N', N'-tetrakis (hydroxyethyl) adipamide (PrimidXL-552 manufactured by Ems-Chemie).

In the present invention, only one type of cross-linking agent may be used alone, or a plurality of cross-linking agents may be used in combination. The amount of the cross-linking agent used may be determined in consideration of the type and the number of moles of the functional group contained in the block polymer (A), and is not particularly limited, but is usually the block polymer (A). It is used in the range of 0.1 parts by mass to 10 parts by mass with respect to 100 parts by mass. Further, if it is blended in a range smaller than the number of moles of the functional group contained in the block polymer (A), the unreacted cross-linking agent is less likely to be released, which is preferable.

<Base layer and surface layer>
As the base material layer of the cell culture member, at least one selected from the group consisting of polyolefin, polycycloolefin, polystyrene, glass, and silicone can be used.

The form of the cell culture member may be a sheet shape, a plate shape, or a dish shape as well as a three-dimensional molded body. Examples of the sheet-like material include films, non-woven fabrics, and papers. Examples of the plate-shaped plate include flat-bottomed plates having 6 holes, 12 holes, 24 holes, 96 holes and the like. Examples of the dish shape include dishes having diameters of 35 mm, 60 mm, 90 mm, 100 mm and the like. As the cell culture member, a silicone film is also preferably used from the viewpoint of oxygen permeability.

The method for forming the surface layer is not particularly limited, but a method of coating the substrate with a coating agent containing the block copolymer (A) is preferable. The coating agent contains a block copolymer (A), a solvent, and optionally a cross-linking agent, and the content of the block copolymer (A) is preferably 0.1 to 10% by mass based on the total amount of the coating agent. More preferably, 5 to 3% by mass. Within this range, it is easy to adjust the average surface roughness Ra of the surface layer to a suitable range.

As the coating method, the spin coating method is preferable from the viewpoint of ease of coating and adjustment of the average surface roughness Ra, but the base material can be coated by various coating methods such as brush, dipping, roller, spray, injection, and coating machine. The solution can also be applied to. The film thickness of the coating film after coating and after removing the solvent is not limited, but is preferably 100 μm or less. 10 μm or less is more preferable, and 5 μm or less is more preferable.
The solvent is not particularly limited as long as it can form a surface layer by coating and does not erode the base material layer, but is not particularly limited, but water, methanol, ethanol, acetone, methyl ethyl ketone (hereinafter referred to as MEK), acetonitrile, dimethylformamide. And the like, organic solvents and the like can be mentioned.

The average surface roughness Ra of the surface layer of the present invention is preferably 10 nm or less, more preferably 5 nm or less, and further preferably 3 nm or less. By setting the average surface roughness of the cell culture member of the present invention to 10 nm or less, adherent cell spheroids having a diameter of 50 to 250 μm can be stably obtained.

In the present invention, the sterilization method is not particularly limited, and examples thereof include high-pressure steam sterilization, ethylene oxide gas sterilization, radiation sterilization such as gamma ray sterilization and electron beam sterilization, and gamma ray sterilization and electron beam sterilization are preferable, and mass production is performed. In this case, gamma-ray sterilization is particularly preferable in terms of radiation permeability.

The absorbed dose of radiation is not particularly limited, but if the absorbed dose is too low, sterilization cannot be ensured, and if it is too high, the cell culture vessel and the coating layer of the coating agent deteriorate. The absorbed dose of radiation in the present invention is preferably 1 kGy or more and 50 kGy or less, and particularly preferably 5 kGy or more and 30 kGy or less. Thereby, sterilization can be imparted while sufficiently maintaining the characteristics of the culture vessel of the present invention.

In the present invention, an adherent cell having no differentiation function refers to a cell that does not have a differentiation function and needs to adhere itself to an appropriate surface for proliferation. Examples of cells having no differentiation function include CHO cells, HEK293 cells, HuH-7 cells, Vero cells, NIH / 3T3 cells and the like.

Further, from the viewpoint of drug discovery screening, it is preferable to use cancer cells as adherent cells having no differentiation function. The cancer cell may be an existing cancer cell line or a primary cancer cell line established using a cancer tissue isolated from a human cancer primary tumor. The type of cancer cell is not particularly limited, and may be any cancer such as liver cancer, kidney cancer, malignant brain cancer, pancreatic cancer, gastric cancer, and lung cancer. In addition, although mainly human-derived ones are used, cancer cells derived from non-human animals (specifically, mice, rats, rabbits, pigs, dogs, monkeys, cows, horses, sheep, chickens, etc.) can also be used. good.

The present invention will be described in more detail with reference to the following examples, but the following examples do not limit the scope of rights of the present invention in any way. In the examples, "parts" and "%" represent "parts by mass" and "% by mass", respectively, Mw means the mass average molecular weight, and Tg means the glass transition temperature.

<Measurement method of mass average molecular weight (Mw)>
For the measurement of Mw, GPC (gel permeation chromatography) "GPC-101" manufactured by Showa Denko Corporation was used. GPC is a liquid chromatography in which a substance dissolved in a solvent (THF; tetrahydrofuran) is separated and quantified according to the difference in molecular size. In the measurement in the present invention, two "KF-805L" (manufactured by Showa Denko: GPC column: 8 mm ID x 300 mm size) are connected in series to the column, and the sample concentration is 1% by mass and the flow rate is 1.0 ml / min. The pressure was 3.8 MPa and the column temperature was 40 ° C., and the mass average molecular weight (Mw) was determined in terms of polystyrene. In the data analysis, the calibration curve, molecular weight, and peak area were calculated using the manufacturer's built-in software, and the mass average molecular weight was obtained with the holding time in the range of 15 to 30 minutes as the analysis target.

<Measurement method of glass transition temperature (Tg)>
For the block copolymer from which the solvent has been dried and removed, a sample amount of about 5 mg was weighed in a standard aluminum container using "DSC-1" manufactured by METTLER TOLEDO, and the temperature modulation amplitude was ± 1 ° C. and the temperature modulation cycle was 60 seconds. The temperature was measured from -80 to 200 ° C. under the condition of a temperature rising rate of 2 ° C./min, and the glass transition temperature was determined from the differential thermal curve of the reversible component.

<Measurement method of average surface roughness Ra>
The average surface roughness Ra (arithmetic mean surface roughness Ra) shown in the present invention is a value represented by the abbreviation of Ra according to JISB0601, and represents the average value of the absolute value deviation from the average line of the surface roughness values.

<Synthesis of block copolymer>
[Manufacturing Example 1]
A reaction vessel equipped with a thermometer, a stirrer, a nitrogen introduction tube, a reflux condenser, and a dropping tube was charged with 130 parts by mass of MEK as an organic solvent under a nitrogen stream, and heated at 80 ° C. for 30 minutes under stirring. Add 100 parts by mass of methoxyethyl acrylate as a monomer, 4.2 parts by mass of V501 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd .: azo initiator) as a polymerization initiator, and 10 parts by mass of MEK as a solvent in a dropping tube for 2 hours. Dropped over. After aging for 5 hours after completion of the dropping, the mixture was cooled to room temperature to stop the reaction, and an acrylic resin having a carboxylic acid terminal was obtained. After that, 1 part of PEG2000 (manufactured by Nichiyu Co., Ltd., polyethylene glycol, Mn = 2000, hydroxyl value = 56) and 0.05 part of tetrabutyl orthotitanate as a catalyst were charged, and the temperature was raised to 85 ° C., and MEK flowed out. After confirming, the temperature was raised to 120 ° C., and then the dehydration reaction was continued while raising the temperature by 10 ° C. every 30 minutes. After the temperature reached 230 ° C., the reaction was continued at the same temperature for 3 hours, held for 1 hour with a vacuum of about 2 kPa, and further reacted for 2 to 3 hours with a vacuum of about 1 kPa to lower the temperature. When the internal temperature was lowered to 100 ° C., a mixed solvent consisting of ethanol / water = 90/10 parts by mass was added and diluted to obtain a block polymer solution having a solid content of 1% by mass. The obtained block polymer had a Mw of 45,000, a Tg of −43 ° C., a PEG ratio of 0.95% by mass, and a monomer average LogP of 0.48.
[Manufacturing example 2]
In a reaction vessel equipped with a thermometer, a stirrer, a nitrogen introduction tube, a reflux condenser, and a dropping tube, 130 parts by mass of acetone was charged as an organic solvent under a nitrogen stream and heated at 55 ° C. for 30 minutes under stirring. Add 100 parts by mass of tetrahydrofurfuryl acrylate as a monomer, 3.9 parts by mass of VA086 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd .: azo initiator) as a polymerization initiator, and 10 parts by mass of acetone as a solvent in the dropping tube. Dropped over time. After aging for 5 hours after completion of the dropping, the mixture was cooled to room temperature to stop the reaction, and an acrylic resin having a hydroxyl group at the end was obtained. Then, 0.5 part of PEG2000 (manufactured by Nichiyu Co., Ltd., polyethylene glycol, Mn = 2000, hydroxyl value = 56), 0.5 part of isophorone diisocyanate, 0.01 part of dimethylbenzylamine as a catalyst were charged, and the temperature was 55 ° C. The temperature was raised to FT-IR, and the reaction was carried out until the peak derived from the isocyanate group (around 2254 cm-1) disappeared by FT-IR. After the reaction was stopped, acetone was removed with a diaphragm pump, and a mixed solvent consisting of ethanol / water = 90/10 parts by mass was added for dilution to obtain a block polymer solution having a solid content of 1% by mass. The obtained block polymer had a Mw of 51,000, a Tg of −10 ° C., a PEG ratio of 0.48% by mass, and a monomer average LogP of 0.78.
[Manufacturing Example 3]
In a reaction vessel equipped with a thermometer, a stirrer, a nitrogen introduction tube, a reflux condenser, and a dropping tube, 130 parts by mass of acetone was charged as an organic solvent under a nitrogen stream and heated at 55 ° C. for 30 minutes under stirring. Add 100 parts by mass of tetrahydrofurfuryl acrylate as a monomer, 7.0 parts by mass of VA057 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd .: azo initiator) as a polymerization initiator, and 10 parts by mass of acetone as a solvent in the dropping tube. Dropped over time. After aging for 5 hours after the completion of the dropping, the mixture was cooled to room temperature to stop the reaction, and an acrylic resin having a carboxyl group at the end was obtained. After that, 1 part of EX861 (manufactured by Nagase ChemteX Corporation, polyethylene glycol diglycidyl ether, epoxy equivalent = 551 g / eq.) Was charged, 0.01 part of dimethylbenzylamine was charged as a catalyst, the temperature was raised to 55 ° C., and acetone was added. It reacted while distilling off. After the reaction was stopped, acetone was completely removed with a diaphragm pump, and a mixed solvent consisting of ethanol / water = 90/10 parts by mass was added for dilution to obtain a block polymer solution having a solid content of 1% by mass. The obtained block polymer had a Mw of 39,000, a Tg of −8 ° C., a PEG ratio of 0.93% by mass, and a monomer average LogP of 0.78.
[Manufacturing Example 4]
A reaction vessel equipped with a thermometer, a stirrer, a nitrogen introduction tube, a reflux condenser, and a dropping tube was charged with 65 parts by mass of MEK as an organic solvent under a nitrogen stream, and heated at 80 ° C. for 30 minutes under stirring. 50 parts by mass of methoxyethyl acrylate as a monomer, 0.475 parts by mass of VPE0201 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd .: Macroazo initiator) as a polymerization initiator, and 10 parts by mass of MEK as a solvent are charged in a dropping tube for 2 hours. Dropped over. After aging for 5 hours after the completion of the dropping, the mixture was cooled to room temperature and the reaction was stopped. Then, after removing MEK with a diaphragm pump, a mixed solvent consisting of ethanol / water = 90/10 parts by mass was added and diluted to obtain a block polymer solution having a solid content of 1% by mass. The Mw of the obtained block copolymer was 125,000, the Tg was −45 ° C., the proportion of PEG was 0.94% by mass, and the average LogP of the monomers was 0.48.

[Production Examples 5 to 12, Comparative Production Examples 1 to 4]
Synthesis was carried out in the same manner as in Production Example 4 according to the composition in Table 1 and the mass parts charged. The characteristic values are shown in Table 1.

In Table 1, the abbreviations are as follows.
MEA: Methoxyethyl acrylate, LogP = 0.48
THFA: Tetrahydrofurfuryl Acrylate, LogP = 0.78
ISTA: Isostearyl acrylate, LogP = 6.00
St: Styrene, LogP = 2.70
GLM: Glycerin monomethacrylate, LogP = -0.06
HEA: 2-hydroxyethyl acrylate, LogP = 0.12
BA: Butyl acrylate, LogP = 1.88
AA: Acrylic acid, LogP = 0.38
ADE200: Polyethylene glycol diacrylate VPE0201: Polyethylene glycol unit-containing polymer azo polymerization initiator AIBN: 2,2'-azobisisobutyronitrile

[Examples 1 to 21], [Comparative Examples 1 to 4]
A curing agent diluted to 1% by mass with each block polymer solution having a solid content of 1% and a mixed solvent consisting of ethanol / water = 90/10 parts by mass according to Production Examples 1 to 12 and Comparative Production Examples 1 to 4. The coating solution was prepared according to the charged mass portion in Table 2. For cell culture members coated with this coating solution, the following are the average surface roughness, solubility in water, cytotoxicity, spheroid formation, spheroid size uniformity, presence or absence of internal necrosis of spheroids, and during medium replacement. The presence or absence of spheroid removal and the processability of the coating film were evaluated. The results are shown in Table 2.

In Table 2, the abbreviations are as follows.
IPDI: Isophorone diisocyanate TC400: Matsumoto Fine Chemical Co., Ltd., Titanium chelate compound V02: Nisshinbo Co., Ltd., Aqueous carbodiimide group-containing compound EX521: Nagase ChemteX Co., Ltd., Polyfunctional aliphatic epoxy compound ALCH: Kawaken Fine Chemical Co., Ltd., Aluminum chelate Compound Chemitite PZ: Polyfunctional azilysin compound XL552 manufactured by Nippon Catalyst Co., Ltd., hydroxyalkylamide group-containing compound manufactured by MSM.

<Evaluation>
(1) Average surface roughness Ra
To an untreated dish (manufactured by Corning, material: polystyrene) having a diameter of 35 mm, 1 mL of the block polymer solution (coating agent) of the production example was added, and spin coating was performed at 3000 rpm for 30 seconds. After drying at room temperature for 1 hour, the dish was dried at 50 ° C. for 3 hours to prepare a dish whose inner surface was coated with a coating agent. The average surface roughness of this surface was measured by a white light interference type surface texture measuring machine.

(2) Solubility in water About 2 g of the block polymer solution (coating agent) of the production example was added to a precision-weighed untreated dish (made by Corning, material: polystyrene) with a diameter of 35 mm, and the temperature was 50 ° C. for 8 hours. It was dry. After taking out from the oven and precisely weighing the dish, 3 g of ion-exchanged water was added to the dish and the dish was allowed to stand overnight. After the ion-exchanged water was sucked and discharged from the shallow metal container, it was dried again at 50 ° C. for 8 hours, and the shallow metal container was precisely weighed. Solubility in water was calculated by the following formula, and solubility in water was evaluated based on a three-step evaluation standard.
Solubility in water = 100-[(zx) / (yx)] x 100
x: Mass of dish y: Mass before treatment with ion-exchanged water z: Mass after treatment with ion-exchanged water a: Solubility in water ≤ 5%
b: 5% <solubility in water ≤ 50%
c: 50% <solubility in water

(3) Cytotoxicity In a U-shaped bottom 96-well plate, about 0.5 mL of the block polymer solution of the production example was injected into each well. After sucking and discharging this, the plate was dried at 50 ° C. for 3 hours to prepare a plate whose inner surface was coated with a block polymer.
After sterilizing this with ethylene oxide gas, 10% of fetal bovine serum (FBS) was added to Dalveco's modified Eagle's medium (DMEM) as the medium, and a cell line for mouse fibroblasts (NIH / 3T3 cells) was added per well. 1 × seeded 10 ^4/100 [mu] L, and cultured to 5 days in 5% CO _2/37 ℃ incubator.
Cytotoxicity was assessed by performing an ATP assay after 1, 2.5 days. Specifically, 100 μL of ATP reagent (“lump” ATP measurement reagent: manufactured by Toyo Venet Co., Ltd.) is added to the well after culturing, pipetting 5 times, allowing to stand at room temperature for 5 minutes, and then 100 mL of reagent. -The cell lysate was separated into a separate plate and stirred for 1 minute. The amount of light emitted from this was measured using MisrasLB940 (manufactured by Berthold).
Cytotoxicity = (luminance after 5 days of culture) / (luminance after 1 day of culture) x 100
a: 80% ≤ cytotoxicity b: 20% ≤ cytotoxicity <80%
c: Cytotoxicity <20%

(4) Formability of spheroids 1 mL of the block polymer solution of the production example was added to an untreated dish (manufactured by Corning, material: polystyrene) having a diameter of 3.5 cm, and spin-coated at 3000 rpm for 30 seconds. A dish whose inner surface was coated with a coating agent was prepared by drying at room temperature for 1 hour and then at 50 ° C. for 3 hours. After sterilizing this with ethylene oxide gas, 10% of fetal bovine serum (FBS) was added to Dulbecco's modified Eagle's medium (DMEM) as the medium, and a cell line for mouse fibroblasts (NIH / 3T3 cells) was added per dish. 1.5 × 10 ⁵ cells / 2 mL were seeded and cultured to day 3 with 5% _CO 2/37 ℃ incubator.
The spheroid-forming property was determined by the following criteria by photographing the cell culture state with a transmission optical microscope at 40 times after 3 days and observing the cell morphology.
a: 5 or more spheroids are observed per field of view b: 2 or more and less than 5 spheroids are observed per field of view c: 1 spheroid is observed per field of view d: Spheroids are observed in the field of view However, cell adhesion and extension are observed.

(5) Uniformity of spheroid size Using the dish judged as a or b in (4) above, the cell culture state after 3 days was photographed with a transmission optical microscope at 40 times, and the cell morphology was observed. Evaluated by doing.
a: The diameter of the formed spheroid is in the range of 50 to 250 μm b: The diameter of the formed spheroid is outside the range of 50 to 250 μm.

(6) Presence or absence of internal necrosis of spheroids Celstain cells commercially available from Dojin Chemical Research Institute Co., Ltd. after further culturing the dish judged as a or b in (4) above for another 24 hours. The presence or absence of internal necrosis was evaluated by staining using a double staining kit and observing with a fluorescence microscope.
The Celstain Cell Double Stain Kit is a combination of the live cell staining fluorescent dye Calcein-AM and the dead cell staining fluorescent dye PI (Propidium Iodide), and stains live and dead cells in green and red, respectively. Can be done.
The presence or absence of internal necrosis was determined according to the following criteria.
a: The central part of the formed spheroid is dyed green b: The central part of the formed spheroid is dyed red

(7) Presence or absence of spheroid removal during medium replacement 1 mL of the block polymer solution of the production example was added to an untreated dish (manufactured by Corning, material: polystyrene) having a diameter of 3.5 cm, and spin-coated at 3000 rpm for 30 seconds. A dish whose inner surface was coated with a coating agent was prepared by drying at room temperature for 1 hour and then at 50 ° C. for 3 hours. After sterilizing this with ethylene oxide gas, 10% of fetal bovine serum (FBS) was added to Dulbecco's modified Eagle's medium (DMEM) as the medium, and a cell line for mouse fibroblasts (NIH / 3T3 cells) was added per dish. 1.5 × 10 ⁵ cells / 2 mL were seeded and cultured to day 3 with 5% _CO 2/37 ℃ incubator.
Then, in the medium exchange, 50% of the medium in the dish was aspirated, and then the same amount of fresh medium was injected. After repeating this twice, whether or not the spheroid was present on the dish was photographed with a transmission optical microscope at 40 times and judged according to the following criteria.
a: Spheroids are observed b: Spheroids are not observed

(4)'Spheroids formed after gamma sterilization 1 mL of the block polymer solution of the production example was added to an untreated dish (manufactured by Corning, material: polystyrene) having a diameter of 3.5 cm, and spin-coated at 3000 rpm for 30 seconds. A dish whose inner surface was coated with a coating agent was prepared by drying at room temperature for 1 hour and then at 50 ° C. for 3 hours. This is vacuum-wrapped in a transparent film, sterilized by irradiating with gamma rays at 10 kGy, and then using 10% of fetal bovine serum (FBS) added to Dalveco's modified Eagle's medium (DMEM) as a medium for mouse fibroblasts. cell lines (NIH / 3T3 cells) 1.5 × 10 ⁵ cells per dish / 2 mL were seeded and cultured to day 3 with 5% CO _2/37 ℃ incubator.
The spheroid-forming property was determined by the following criteria by photographing the cell culture state with a transmission optical microscope at 40 times after 3 days and observing the cell morphology.
a: 5 or more spheroids are observed per field of view b: 2 or more and less than 5 spheroids are observed per field of view c: 1 spheroid is observed per field of view d: Spheroids are observed in the field of view However, cell adhesion and extension are observed.

(5) Uniform size of spheroids after gamma-ray sterilization Using the dish judged as a or b in (4) above, the cell culture state after 3 days was photographed with a transmission optical microscope at 40 times. , Evaluated by observing cell morphology.
a: The diameter of the formed spheroid is in the range of 50 to 250 μm b: The diameter of the formed spheroid is outside the range of 50 to 250 μm.

(6)'Presence or absence of internal necrosis of spheroids after gamma-ray sterilization The spheroids after culturing the dish judged as a or b in the above (4)' for another 24 hours are commercially available from Dojin Chemical Laboratory Co., Ltd. The presence or absence of internal necrosis was evaluated by staining using the cell stain cell double staining kit and observing with a fluorescence microscope.
The Celstain Cell Double Stain Kit is a combination of the live cell staining fluorescent dye Calcein-AM and the dead cell staining fluorescent dye PI (Propidium Iodide), and stains live and dead cells in green and red, respectively. Can be done.
The presence or absence of internal necrosis was determined according to the following criteria.
a: The central part of the formed spheroid is dyed green b: The central part of the formed spheroid is dyed red

(7)'Presence or absence of spheroid removal when exchanging medium after gamma ray sterilization Add 1 mL of the block polymer solution of the production example to an untreated dish (made by Corning, material: polystyrene) with a diameter of 3.5 cm, and add 1 mL to it at 3000 rpm for 30 seconds. Spin coated. A dish whose inner surface was coated with a coating agent was prepared by drying at room temperature for 1 hour and then at 50 ° C. for 3 hours. This is vacuum-wrapped in a transparent film, sterilized by irradiating with gamma rays at 10 kGy, and then using 10% of fetal bovine serum (FBS) added to Dalveco's modified Eagle's medium (DMEM) as a medium for mouse fibroblasts. cell lines (NIH / 3T3 cells) 1.5 × 10 ⁵ cells per dish / 2 mL were seeded and cultured to day 3 with 5% CO _2/37 ℃ incubator.
Then, in the medium exchange, 50% of the medium in the dish was aspirated, and then the same amount of fresh medium was injected. After repeating this twice, whether or not the spheroid was present on the dish was photographed with a transmission optical microscope at 40 times and judged according to the following criteria.
a: Spheroids are observed b: Spheroids are not observed

(8) Workability of coating film The coating agent prepared in Examples and Comparative Examples is applied to a polyethylene terephthalate (PET) film so that the film thickness after drying is 5 to 10 μm, and dried at 100 ° C. for 2 minutes. After that, it was aged at 50 ° C. for 3 hours. When cutting this coating film, the workability was evaluated based on whether or not the coating film adhered to the blade. The processability of the coating film was evaluated based on the following three-stage evaluation criteria.
a: No coating film adheres to the blade b: Slight coating film adheres to the blade c: The coating film adheres severely to the blade, which poses a practical problem

As can be seen from Examples and Comparative Examples shown in Table 2, the block polymer (A) having an acrylic polymer moiety (A1) and a polyethylene oxide moiety (A2) used in Examples 1 to 21 is a block polymer (A). Compared to the polymers used in Comparative Examples 1 to 4, the polymer has an acrylic polymer moiety composed of a monomer having an average value of LogP of 0 to 2, and the mass% of the polyethylene oxide moiety (A2) is 0. Since it is 01 or more and less than 1, solubility in water is suppressed, and it is possible to obtain physical properties that satisfy all of cytotoxicity, spheroid formation property, size uniformity, internal necrosis state, and spheroid removal property at the time of medium exchange. did it. In addition, spheroids could be formed even after gamma-ray sterilization, and the size uniformity, internal necrosis state, and spheroid removal property at the time of medium replacement were also satisfied.

On the other hand, as in Comparative Example 1, even if the main chain has an acrylic polymer moiety composed of a monomer having an average LogP value of 0 to 2, the polyethylene oxide moiety (A2) is 1% by mass or more. In addition, size non-uniformity and internal necrosis due to aggregation of spheroids were observed. Further, as in Comparative Example 4, even if the main chain has an acrylic polymer portion composed of a monomer having an average value of LogP of 0 to 2, if the polyethylene oxide portion (A2) is not adhered, the spheroid is used. Was not formed, and elongation and proliferation due to cell adhesion were observed.
Further, in Examples 12, 14, 16 to 20, by using a cross-linking agent in combination, it was possible to impart better processability of the coating film as a coating film.

The block copolymer (A) of the present invention can be used as a cell culture member for drug discovery research and regenerative medicine research. When cells are cultured on this cell culture member, spheroids can be formed in an appropriate size, so that they can be developed in applications such as drug discovery screening.

Claims (9)

A cell culture member having a base material layer and a surface layer for culturing adherent cell spheroids having a diameter of 50 to 250 μm and having no differentiation function, and satisfying the following (1) and (2). Material.
(1) The surface layer has an acrylic polymer portion (A1) formed from an acrylic monomer having an average value of water / 1-octanol partitioning coefficient (LogP) of 0 or more and 2 or less, and a polyethylene oxide portion (A2). ) Contains a block polymer (A) formed by bonding via any of the structures of the following formulas (IA) to (IC), and the block polymer (A) is the mass of the polyethylene oxide moiety (A2). / [Total mass of acrylic polymer moiety (A1) and polyethylene oxide moiety (A2)] is 0.01% or more and less than 1%.

(2) The base material layer contains at least one selected from the group consisting of polyolefin, polycycloolefin, polystyrene, glass, and silicone. The cell culture member according to claim 1, wherein the average surface roughness Ra of the surface layer is 10 nm or less. The block polymer (A) uses a polymer azo-based polymerization initiator represented by the following general formula (II), which has a polyethylene oxide moiety (A2) and a mass average molecular weight of 5,000 to 100,000. The cell culture member according to claim 1 or 2, which is a block copolymer obtained by polymerizing an acrylic monomer.

In the formula, m and n represent integers of 1 or more. The cell culture member according to any one of claims 1 to 3, wherein the block polymer (A) has a Tg of −70 ° C. to 70 ° C. and contains a C element, an H element, and an O element in a side chain. The cell culture member according to any one of claims 1 to 4, which is a dish. The cell culture member according to any one of claims 1 to 5, which is used for drug discovery screening. A method for producing a cell culture member having a base material layer and a surface layer for culturing adherent cell spheroids having a diameter of 50 to 250 μm and having no differentiation function.
The acrylic polymer moiety (A1) formed from an acrylic monomer having an average value of water / 1-octanol partitioning coefficient (LogP) of 0 or more and 2 or less and the polyethylene oxide moiety (A2) are represented by the following formula (IA). ) To (IC), which is a block polymer (A) bonded via any of the structures, and the mass of the polyethylene oxide moiety (A2) / [acrylic polymer moiety (A1) and polyethylene oxide moiety (A1). A cell in which the base material layer is coated with a coating agent containing 0.1 to 10% by mass of the block polymer (A) having a total mass with A2) of 0.01% or more and less than 1% to form a surface layer. A method for manufacturing a member for culturing.

The method for producing a cell culture member according to claim 7, further comprising a step of sterilizing with gamma rays. The method for producing a cell culture member according to claim 7 or 8, wherein the method of coating the base material layer with the coating agent is a spin coating method.

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