JP2019180274A - Cell culture appliance processing agent, cell culture appliance, medical device and production method of cell culture appliance - Google Patents

Abstract

To provide a cell culture appliance processing agent in which protein absorption is suppressed, excellent cell adhesion prevention effect can be achieved, and which can exhibit adhesion to a hydrophobic material and transparency, a cell culture appliance suitable for production of a cell lump and having a cell adhesion prevention film formed of the cell culture appliance processing agent, a medical device having the appliance, and a production method of the cell culture appliance.SOLUTION: The above-mentioned problem can be solved by a cell culture appliance processing agent including an urethane based polymer (A1) including at least any betaine structure indicated by one of the general formulae 1-3. The problem can be solved by a cell culture appliance processing agent including a vinyl-based polymer (A2) having on a side chain, at least any betaine structure indicated by the general formula 4 or 5.SELECTED DRAWING: None

Description

  The present invention relates to a cell culture equipment treatment agent, a cell culture equipment, a medical device, and a method for producing a cell culture equipment.

  Evaluation using cells is widely performed in the fields of drug discovery such as pharmaceuticals and agricultural chemicals, food inspection, diagnosis, and genetic engineering. Conventionally, when evaluating the function and response of a cell, the cell is cultured in a medium or the like placed in a petri dish or the like and used for various experiments. However, when investigating reactions caused by a large number of chemicals and introduced substances, there is a demand for an evaluation method that can perform specific types of cell reactions efficiently, perform various types of analysis in a short time, and obtain results with fewer samples. It was. Under such circumstances, an attempt to observe or measure the chemical reaction of cells held on a substrate such as glass by a cell culture device called a microchip brings advantages such as simplified operation and reduced analysis time. In recent years, it has been actively performed. As an advantage of a system using a microchip, it is considered to realize an inexpensive system that can be carried in a small space by reducing the amount of samples and reagents used or discharging waste liquid. For example, Non-Patent Document 1 makes use of the above-described advantages and studies to evaluate the function and response of a cell using a microchip in which cells are attached to the channel wall surface.

  However, in the microchip using cells, there is a problem that adhesion due to cells or proteins derived from biological samples occurs on the surface of the device, leading to clogging and a decrease in accuracy and sensitivity of analysis. In order to solve the problem, Patent Document 1 includes a polymer having a hydrophilic repeating unit and a repeating unit having a polyoxyalkylene group in a side chain and a specific group at the end of the side chain. Cell adhesion inhibitors are described, and specific betaine structures are disclosed as hydrophilic repeating units. However, since most of the microchips are hydrophobic materials such as silicone, polyolefin, polystyrene, etc., the cell adhesion inhibitor of Patent Document 1 having high hydrophilicity is insufficient in wettability to a hydrophobic substrate and has poor adhesion. It was enough. Moreover, there was a problem that the transparency of the flow path device was inferior.

International Publication No. 2017/022815

Analytical Chemistry 2005: 77, p. 215-2131

The present invention relates to a cell culture equipment treating agent that suppresses protein adsorption, has an excellent cell adhesion preventing effect, and exhibits adhesion and transparency to a hydrophobic substrate, and the cell culture equipment treating agent. An object of the present invention is to provide a cell culture device suitable for cell mass production, having a cell adhesion prevention film, a medical device comprising the device, and a method for producing the cell culture device.
That is, the present invention relates to <1> to <10>.

<1> A cell culture equipment treating agent comprising a urethane polymer (A1) having at least one of the structures represented by the following general formulas 1 to 3.
General formula 1

General formula 2

General formula 3

(In general formulas 1 to 3,
R ¹ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
R ² , R ⁴ and R ⁷ are each independently an alkylene group having 1 to 6 carbon atoms,
R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ are each independently an alkyl group, aryl group, aralkyl group, or pyridyl group,
Y is -COO ^- or -SO ₃ ^-,
X is an oxygen atom or -NH-,
* Represents the bonding position with the main chain of the urethane-based polymer. )

<2> A cell culture equipment treating agent comprising a vinyl polymer (A2) having at least one structure represented by the following general formula 4 or 5 in a side chain.
Formula 4

Formula 5

(In general formulas 4 and 5,
Y is -COO ^- or -SO ₃ ^-,
4 of R ^{10 to} R ¹⁴ are a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
One of R ^{10 to} R ¹⁴ is a bonding position with the main chain of the vinyl polymer,
R ¹⁵ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
R ¹⁶ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ¹⁷ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
* Represents a bonding position with the main chain of the vinyl polymer. )

<3> The cell culture equipment treatment according to <1>, wherein the urethane-based polymer (A1) includes at least one of the structures represented by the general formulas 1 to 3 in a total amount of 0.25 to 1.5 mmol / g. Agent.

<4> The cell culture equipment treating agent according to <2>, wherein the vinyl polymer (A2) has at least one crosslinkable group selected from the group consisting of a carboxyl group and a hydroxyl group.

<5> The cell culture equipment treatment agent according to any one of <1> to <4>, further comprising a crosslinking agent.

<6> A cell culture equipment having a cell adhesion prevention film formed from the cell culture equipment treatment agent according to any one of <1> to <5> on a base material.

<7> The cell culture device according to <6>, wherein the base material is a silicone base material.

<8> The cell culture device according to <6> or <7>, wherein the substrate is a silicone substrate having a microchannel.

<9> A method for producing cell culture equipment, comprising a step of forming a cell adhesion prevention film formed from the cell culture equipment treatment agent according to any one of <1> to <5> on a base material.

<10> A medical device comprising the cell culture device according to any one of <6> to <8>.

  According to the present invention, it is possible to provide a cell culture equipment treating agent having an excellent protein adhesion preventing effect, adhesion to a hydrophobic substrate, and high transparency, and to easily produce a cell mass. It is possible to provide a highly transparent cell culture device, a medical device, and a method for producing the cell culture device that can observe the behavior of cells visually.

The cell culture equipment treating agent of the present invention comprises a urethane polymer (A) having at least one of the structures represented by the general formulas 1 to 3. The cell culture equipment treating agent of the present invention is characterized by containing a vinyl polymer (B) having at least one of the structures represented by the general formula 4 or 5 in the side chain.
The general formulas 1 to 5 all have a betaine structure. A betaine structure refers to a structure in which a positive charge and a negative charge are not adjacent to each other in the same molecule, and a dissociable hydrogen atom is not bonded to a positively charged atom. By having a betaine structure in the molecule, there is an excellent effect of preventing protein adhesion.

<Cell culture equipment treatment agent>
<Urethane-based polymer (A1)>
The urethane polymer (A1) in the present invention has at least one of the structures represented by general formulas 1 to 3. By having at least one of the structures represented by the general formulas 1 to 3, the surface of the hydrophobic base material is well wetted, and the transparency and adhesion of the base material are improved.

General formula 1

General formula 2

General formula 3

(In general formulas 1 to 3,
R ¹ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
R ² , R ⁴ and R ⁷ are each independently an alkylene group having 1 to 6 carbon atoms,
R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ are each independently an alkyl group, aryl group, aralkyl group, or pyridyl group,
Y is -COO ^- or -SO ₃ ^-,
X is an oxygen atom or a nitrogen atom,
* Represents the bonding position with the main chain of the urethane-based polymer. )

  The urethane-based polymer (A1) in the present invention has a polyol component, a polyisocyanate component, and a tertiary amino group-containing monomer (hereinafter also referred to as a polyol having a tertiary amino group) as an introduction source of a betaine structure as essential components. It is a reaction product with a polyamine component, a monofunctional hydroxyl component or a monofunctional amine component used as necessary.

  In the present invention, the urethane-based polymer containing the structure is polymerized after the tertiary amino group-containing monomer as a precursor for forming the structure is betainized using a betainating agent (B) described later, Alternatively, it may be obtained by any method, for example, by polymerizing a tertiary amino group-containing monomer as a precursor for forming the structure to obtain a polymer having a tertiary amino group and then converting the polymer into a betaine. It may be what was made.

[Tertiary amino group-containing monomer]
Here, examples of the tertiary amino group-containing monomer as a precursor before betaineization include tertiary amino group-containing diols having 1 to 20 carbon atoms. For example, N-alkyl dialkanolamine and N, N-dialkyl monoalkanolamine are mentioned.
Examples of the N-alkyl dialkanolamine include N-methyldiethanolamine, N-ethyldiethanolamine, N-propyldiethanolamine, N-butyldiethanolamine, and N-methyldipropanolamine.
Examples of N, N-dialkylmonoalkanolamines include N, N-dimethylethanolamine.
In addition, N, N-bis (2-hydroxyethyl) benzylamine, N, N-bis (2-hydroxyethyl) cyclohexylamine, diethanol-p-toluidine, diisopropanol-p-toluidine, N, N-bis (2 -Hydroxyethyl) aniline, N, N-bis (2-hydroxypropyl) aniline, N, N-bis (2-hydroxyethyl) -3-chloroaniline, N, N-bis (2-hydroxyethyl) -4- Pyridinecarboxamide, N, N-bis (2-hydroxyethyl) -α-aminopyridine, 1,4-bis (2-hydroxyethyl) piperazine, 3-diethylaminopropane-1,2-diol, 3-dimethylaminopropane -1,2-diol and the like are listed as tertiary amino group-containing monomers.

[Betainizing agent (B)]
The betaine agent (B) is composed of cyclic sulfonic acid ester (B1), ω-halogenated alkylsulfonic acid metal salt (B2), cyclic carboxylic acid ester (B3) and ω-halogenated alkylcarboxylic acid metal salt (B4). Selected from the group consisting of It is a group of compounds used for sulfobetainization or carbobetaineization of the amino group of the above-mentioned tertiary amino group-containing monomer.

  Examples of such cyclic sulfonic acid ester (B1) include 1,2-ethane sultone, 1,3-propane sultone, and 1,4-butane sultone.

Examples of such ω-halogenated alkylsulfonic acid metal salt (B2) include 2-
Examples include sodium chloroethanesulfonate, sodium 2-bromoethanesulfonate, sodium 3-chloropropanesulfonate, sodium 3-bromopropanesulfonate, sodium 4-chlorobutanesulfonate, sodium 4-bromobutanesulfonate, and the like.

  Examples of such cyclic carboxylic acid ester (B3) include β-propiolactone, γ-butyrolactone, δ-valerolactone, and the like.

  Examples of such ω-halogenated alkylcarboxylic acid metal salt (B4) include sodium 2-chloroacetate, sodium 2-bromoacetate, sodium 3-chloropropionate, sodium 3-bromopropionate, 4-chlorobutyric acid. Examples thereof include sodium, sodium 4-bromobutyrate, sodium 5-chloropentanoate, sodium 5-bromopentanoate and the like.

[Polyol component]
The polyol component is not particularly limited, but glycols such as ethylene glycol, 1,4-butanediol, neopentyl glycol, butylethylpentanediol, glycerin, trimethylolpropane, pentaerythritol, polyester polyol, polyether Polyol, polyester polyol, polybutadiene polyol, hydrogenated polybutadiene polyol, polyisoprene polyol, hydrogenated polyisoprene polyol or polyurethane polyol which is a reaction product of polyether polyol and polyisocyanate, polyether adduct of polyhydric alcohol, and the like.

[Polyisocyanate component]
Examples of the polyisocyanate component include aromatic polyisocyanate, aliphatic polyisocyanate, araliphatic polyisocyanate, and alicyclic polyisocyanate.

  Aromatic polyisocyanates include 1,3-phenylene diisocyanate, 4,4′-diphenyl diisocyanate, 1,4-phenylene diisocyanate, 4,4′-diphenylmethane diisocyanate, 2,4-tolylene diisocyanate, 2,6-triisocyanate. Range isocyanate, 4,4'-toluidine diisocyanate, 2,4,6-triisocyanate toluene, 1,3,5-triisocyanate benzene, dianisidine diisocyanate, 4,4'-diphenyl ether diisocyanate, 4,4 ', 4 " -Triphenylmethane triisocyanate etc. can be mentioned.

Aliphatic polyisocyanates include trimethylene diisocyanate, tetramethylene diisocyanate, hexamethylene diisocyanate, pentamethylene diisocyanate, 1,2-propylene diisocyanate, 2,3-butylene diisocyanate, 1,3-butylene diisocyanate, dodecamethylene diisocyanate, 2, Examples include 4,4-trimethylhexamethylene diisocyanate.

  As the aromatic aliphatic polyisocyanate, ω, ω′-diisocyanate-1,3-dimethylbenzene, ω, ω′-diisocyanate-1,4-dimethylbenzene ω, ω′-diisocyanate-1,4-diethylbenzene, 1, Examples include 4-tetramethylxylylene diisocyanate and 1,3-tetramethylxylylene diisocyanate.

Examples of alicyclic polyisocyanates include 3-isocyanate methyl-3,5,5-trimethylcyclohexyl isocyanate, 1,3-cyclopentane diisocyanate, 1,3-cyclohexane diisocyanate, 1,4-cyclohexane diisocyanate, and methyl-2,4. -Cyclohexane diisocyanate, methyl-2,6-cyclohexane diisocyanate, 4,4'-methylenebis (cyclohexyl isocyanate), 1,4-bis (isocyanatemethyl) cyclohexane, 1,4-bis (isocyanatemethyl) cyclohexane, etc. it can.

  Further, a so-called adduct obtained by adding a trifunctional alcohol such as trimethylolpropane to the polyisocyanate, a burette obtained by reacting the polyisocyanate with water, and a polyisocyanate formed with an isocyanurate ring. A monomer or the like can also be used in combination. A reaction product of the aforementioned polyhydric alcohol polyether adduct and diisocyanate can also be used as the polyisocyanate component.

As the polyisocyanate used for obtaining the urethane polymer, 4,4′-diphenylmethane diisocyanate, hexamethylene diisocyanate, 3-isocyanate methyl-3,5,5-trimethylcyclohexyl isocyanate (isophorone diisocyanate) and the like are preferable.

[Polyamine component]
In obtaining a urethane polymer, a polyamine component can be used as necessary. Examples of the polyamine component include ethylenediamine, isophoronediamine, phenylenediamine, 2,2,4-trimethylhexamethylenediamine, tolylenediamine, hydrazine, piperazine, hexamethylenediamine, propylenediamine, dicyclohexylmethane-4,4-diamine, Examples of the diamine include 2-hydroxyethylethylenediamine, di-2-hydroxyethylethylenediamine, di-2-hydroxyethylpropylenediamine, 2-hydroxypropylethylenediamine, and di-2-hydroxypropylethylenediamine. Isophorone diamine and 2,2,4-trimethylhexamethylene diamine are preferable because the reaction is easily controlled and hygienic.
By using a polyamine component, a urea bond having a higher cohesive force than that of a urethane bond is formed, so that high adhesion to the substrate can be obtained.

[Monofunctional hydroxyl component]
There is no particular limitation on the monofunctional hydroxyl component used as one of the terminal terminators when obtaining a urethane polymer, and methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1 -A hexanol, 1-octanol, etc. are mentioned, The 1 type (s) or 2 or more types selected from these groups can be used.

[Monofunctional amine component]
The monofunctional amine component used as one of the terminal terminators as well as the monofunctional hydroxyl component is not particularly limited. Hexylamine, octylamine, diethylamine, dodecylamine, hexadecylamine, octadecylamine, stearylamine, Examples include oleylamine, cyclohexylamine, monoethanolamine, monopropanolamine, diethanolamine, and dipropanolamine.

  In the present invention, the preparation of the urethane-based polymer comprises the polyol component, the polyisocyanate component, and the monomer serving as the introduction source of the betaine structure, which are essential components, the polyamine component, the monofunctional hydroxyl component, and the monofunctional monomer used as necessary. All components of the amine component may be reacted at the same time (one-shot method) or may be reacted sequentially. In order to reliably produce a desired urethane-based polymer as a main product, a sequential reaction in which at least one of these components is sequentially reacted is preferable. When preparing a urethane-type polymer by sequential reaction, the following method is applicable, for example.

  A step of reacting a polyol component, a polyisocyanate component, and a monomer serving as an introduction source of a betaine structure under an excess of the polyisocyanate component to obtain a urethane prepolymer having an isocyanate group at the terminal, and then the urethane prepolymer and the polyamine component. To obtain a polyurethane polyurea having an isocyanate group at the end, and finally reacting the remaining isocyanate group with a monofunctional hydroxyl component and / or a monofunctional amine component.

  In addition, how to advance a sequential reaction is not limited to the method illustrated previously.

The urethane polymer of the present invention may be produced by reacting raw materials in the absence of a solvent or may be produced by reacting in an organic solvent.
Organic solvents include ketone compounds such as acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone, ester compounds such as methyl acetate, ethyl acetate, butyl acetate, ethyl lactate and methoxyethyl acetate, ethers such as diethyl ether and ethylene glycol dimethyl ether. Various solvents such as aromatic compounds such as a series compound, aromatic compounds such as toluene and xylene, aliphatic compounds such as pentane and hexane, and halogenated hydrocarbon compounds such as methylene chloride, chlorobenzene, and chloroform can be used.

  Moreover, a catalyst can be added as needed at the time of synthesis of the urethane polymer, for example, a metal catalyst such as dibutyltin diacetate, dibutyltin dilaurate, dioctyltin dilaurate, dibutyltin dimaleate; 1,8-diaza-bicyclo 3 such as (5,4,0) undecene-7,1,5-diazabicyclo (4,3,0) nonene-5,6-dibutylamino-1,8-diazabicyclo (5,4,0) undecene-7 Examples include tertiary amines; reactive tertiary amines such as triethanolamine, and these may be used alone or in combination of two or more.

The isocyanate group-containing urethane prepolymer is preferably obtained by reacting the above-mentioned polyol component, polyisocyanate component, and monomer serving as a betaine structure introduction source in an organic solvent at 120 ° C. or lower in the presence of a catalyst. It is more preferable to react at 110 ° C. for 1 to 20 hours. When the temperature is higher than 110 ° C., it becomes difficult to control the reaction rate, and it becomes difficult to obtain a urethane prepolymer having a predetermined molecular weight and structure.
The reaction between the isocyanate group and the polyamine component is preferably carried out in an organic solvent at 60 ° C. or lower. If the temperature is higher than that, it becomes difficult to control the reaction rate, and it becomes difficult to obtain a urethane polymer having a predetermined molecular weight and structure.

[Betaine structure content]
The betaine structure itself is rich in hydrophilicity, but the urethane polymer (A1) can be rendered water-insoluble by controlling the amount contained in the urethane polymer (A1). In the present invention, water-insoluble means 1 mass of polymer in 99 g of ion-exchanged water at 25 ° C., and after stirring for 24 hours at 25 ° C., the water is removed and the mass is reduced after the polymer is dried under reduced pressure. Is 5% or less. When the polymer (A1) is water-insoluble, water resistance can be imparted to the coating film and durability can be improved.

  The betaine structure content of the polymer (A1) in the present invention is preferably 0.25 to 1.5 mmol / g, more preferably 0.3, from the viewpoint of ensuring cytotoxicity and wettability to the silicone substrate. It is -1.3 mmol / g, More preferably, it is 0.2-1.2 mmol / g. The betaine structure content can be calculated by an analytical method such as NMR or GC-MS.

[Mass average molecular weight (Mw)]
The mass average molecular weight of the urethane-based polymer (A1) is preferably 2,000 from the viewpoints of the cohesive strength of the coating film and the long-term cell adhesion preventing effect due to suppression of peeling from the coating substrate, and the coating property. Or more, more preferably 2,000 to 10,000,000, more preferably 5,000 or more, still more preferably 10,000 or more, and particularly preferably 100,000 or more. More preferably, it is 500,000 or less.
As the mass average molecular weight of the urethane polymer (A1), a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) is adopted. The measuring device and measurement conditions are basically based on the following condition 1 and are allowed to be set to condition 2 depending on the solubility of the sample. However, depending on the polymer species, an appropriate carrier (eluent) and a column suitable for it may be selected and used. For other matters, refer to JISK7252-1-4: 2008. In addition, it is assumed that the hardly soluble polymer compound is measured at a soluble concentration under the following conditions.
Moreover, when the molecular weight measurement of a urethane type polymer (A1) is difficult, the mass average molecular weight of a betaine-ized precursor polymer can be made into the mass average molecular weight of a urethane type polymer (A1). The mass average molecular weight of the betaened precursor polymer is a value measured in terms of standard polystyrene by gel permeation chromatography (GPC), and the measurement apparatus and measurement conditions are basically based on the following condition 3.

(Condition 1)
Column: TOSOHTSKgelSuperHZM-H,
TOSOHTSKgelSuperHZ4000,
TOSOHTSKgelSuperHZ2000
Carrier using tetrahydrofuran-connected column: Tetrahydrofuran Measurement temperature: 40 ° C
Carrier flow rate: 1.0 ml / min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection amount: 0.1 ml

(Condition 2)
Column: TosohtskegelSuperAWM-H Two carriers: 10 mM LiBr / N-methylpyrrolidone Measurement temperature: 40 ° C.
Carrier flow rate: 1.0 ml / min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection amount: 0.1 ml

(Condition 3)
Column: TOSOHTSKgelSuperAW4000,
TOSOHTSKgelSuperAW3000,
TOSOHTSKgelSuperAW2500
Use a column connected to
Carrier: N, N-dimethylformamide (1 L), triethylamine (3.04 g), mixed solution of LiBr (0.87 g) Measurement temperature: 40 ° C.
Carrier flow rate: 0.6ml / min

<Vinyl polymer (A2)>
The vinyl polymer (A2) in the present invention has at least one structure represented by the general formula 4 or 5. By having at least one of the structures represented by the general formula 4 or 5, the surface of the hydrophobic substrate is well wetted, and there is an effect of improving the transparency and adhesion of the substrate. The vinyl polymer (A2) may be referred to as a vinyl polymer.

Formula 4

Formula 5

(In general formulas 4 and 5,
Y is -COO ^- or -SO ₃ ^-,
4 of R ^{10 to} R ¹⁴ are a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
One of R ^{10 to} R ¹⁴ is a bonding position with the main chain of the vinyl polymer,
R ¹⁵ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
R ¹⁶ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ¹⁷ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
* Represents a bonding position with the main chain of the vinyl polymer. )

In the present invention, examples of the vinyl polymer having the structure represented by the general formula 4 or 5 in the side chain include the following two.
(I) Monomer having at least one structure represented by the general formula 4 or 5 (however, the bonding position with the main chain of the vinyl polymer in the general formula 4 or 5 represents the bonding position with the monomer) And a vinyl polymer obtained by polymerizing other monomers.
(II) A polymer which is a reaction product of a vinyl polymer having a structure of at least any tertiary amino group represented by the following general formula 6 or 7 in the side chain and a betaine agent. Specifically, a monomer having a structure of at least any tertiary amino group represented by the following general formula 6 or 7 (provided that the bonding position with the main chain of the vinyl polymer in the general formula 6 or 7 is a monomer) Is a reaction product of a vinyl polymer obtained by polymerizing other monomers and a betaine agent.
The reaction of a tertiary amino group with a betaine agent is hereinafter also referred to as “betaine formation”.

General formula 6

Formula 7

(In general formulas 6 and 7,
Y is -COO ^- or -SO ₃ ^-,
4 of R ^{10 to} R ¹ are a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
One of R ^{10 to} R ¹⁴ is a bonding position with the main chain of the vinyl polymer,
R ¹⁶ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
* Represents a bonding position with the main chain of the vinyl polymer. )

  Although the monomer which comprises the vinyl polymer (A2) of this invention is demonstrated below, it is not limited to these. In the present invention, “(meth) acryl” means both “acryl” and “methacryl”.

<Monomer having a structure represented by Formula 4>
Examples of the monomer having a betaine structure represented by the general formula 4 include 2-vinyl-1- (3-sulfopropyl) pyridinium inner salt, 2-vinyl-1- (3-sulfobutyl) pyridinium inner salt, and the like. 4 such as 2-vinyl-1- (3-sulfoalkyl) pyridinium inner salt; 4-vinyl-1- (3-sulfopropyl) pyridinium inner salt, 4-vinyl-1- (3-sulfobutyl) pyridinium inner salt, etc. -Vinyl-1- (3-sulfoalkyl) pyridinium inner salt, and the like.

<Monomer having structure represented by general formula 5>
Examples of the monomer having a betaine structure represented by general formula 5 include 1-vinyl-3- (3-sulfopropyl) imidazolium inner salt, 1-vinyl-3- (3-sulfobutyl) imidazolium inner salt, 1-vinyl-2-alkyl- such as 1-vinyl-2-methyl-3- (3-sulfopropyl) imidazolium inner salt, 1-vinyl-2-methyl-3- (4-sulfobutyl) imidazolium inner salt 3- (4-sulfoalkyl) imidazolium inner salt, and the like.

<Monomer having structure represented by general formula 6>
Examples of the monomer having a betaine structure represented by the general formula 6 include vinylpyridines such as 4-vinyl-pyridine and 2-vinyl-pyridine.

<Monomer having structure represented by general formula 7>
Examples of the monomer having a betaine structure represented by the general formula 7 include 1-vinyl-2-alkyl-imidazole such as 1-vinylimidazole and 1-vinyl-2-methyl-imidazole.

[Monomer (r) having a crosslinkable group]
The vinyl polymer (A2) preferably has at least one crosslinkable group selected from the group consisting of a carboxyl group and a hydroxyl group from the viewpoint of improving durability. Specifically, when obtaining the vinyl polymer (A2), in addition to the vinyl polymer (A2), a monomer (r) having at least one crosslinkable group selected from the group consisting of a carboxyl group and a hydroxyl group is used. It is preferable to use it. The crosslinkable group derived from the monomer (r) introduces a crosslinked structure into the polymer coating film by reacting with a crosslinking agent described later, and exhibits excellent durability.
For example, a copolymer having a carboxyl group introduced therein can be crosslinked with an epoxy compound, an aziridine compound, an oxetane, a carbodiimide compound, an amino compound, an isocyanate compound, or the like. A copolymer into which a hydroxyl group has been introduced can be crosslinked with a carbodiimide compound, an isocyanate compound, an amino compound, an alkoxylane compound, or the like.

  The carboxyl group-containing monomer is not particularly limited as long as it has a carboxyl group in its structure. For example, (meth) acrylic acid, 2-carboxyethyl (meth) acrylate, or alkylene such as ethylene oxide or propylene oxide Examples include alkylene oxide addition succinic acid (meth) acrylate having a carboxyl group at the terminal where oxide is repeatedly added.

  The hydroxyl group-containing monomer is not particularly limited as long as it has a hydroxyl group in its structure. For example, 2-hydroxyethyl (meth) acrylate, 1-hydroxypropyl (meth) acrylate, (meth) acrylic acid 2-hydroxypropyl, 3-hydroxypropyl (meth) acrylate, 1-hydroxybutyl (meth) acrylate, glycerol monofunctional (meth) acrylate, polylactones having a hydroxyl group at the terminal by ring-opening addition of a lactone ring (meta ) An alkylene oxide addition system (meth) acrylate ester and glucose ring system (meth) acrylate ester having a hydroxyl group at the terminal to which an alkylene oxide such as an acrylic ester, ethylene oxide or propylene oxide is repeatedly added.

  From the viewpoint of durability, the vinyl polymer (A2) preferably has a carboxyl group as a crosslinkable group, and a carboxyl group-containing monomer is preferably used as the monomer (r) having a crosslinkable group constituting the polymer.

<Other monomers>
In obtaining a vinyl polymer, other monomers having one ethylenically unsaturated group in one molecule can be used in addition to the monomer. In addition, by introducing a structure based on a monomer, the polarity and Tg are appropriately controlled, and various physical properties such as excellent coating properties and water resistance can be imparted and controlled.

Examples of other monomers having one ethylenically unsaturated group in one molecule include methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, and pentyl (meth) acrylate. Alkyl (meth) acrylates such as, heptyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, octyl (meth) acrylate, nonyl (meth) acrylate, decyl (meth) acrylate, dodecyl (meth) acrylate;
Α-olefin-based ethylenically unsaturated monomers such as 1-propylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-nonene and 1-decene.
Monomers having a sulfonic acid group such as vinyl sulfonic acid and styrene sulfonic acid;
A monomer having a phosphate group such as (2-hydroxyethyl) methacrylate acid phosphate;
(Meth) acrylamide, N-vinyl-2-pyrrolidone, N-methoxymethyl- (meth) acrylamide, N-ethoxymethyl- (meth) acrylamide, N-propoxymethyl- (meth) acrylamide, N-butoxymethyl- (meta ) Acrylamide, N-pentoxymethyl- (meth) acrylamide, N, N-di (methoxymethyl) acrylamide, N-ethoxymethyl-N-methoxymethylmethacrylamide, N, N-di (ethoxymethyl) acrylamide, N- Ethoxymethyl-N-propoxymethyl methacrylamide, N, N-di (propoxymethyl) acrylamide, N-butoxymethyl-N- (propoxymethyl) methacrylamide, N, N-di (butoxymethyl) acrylamide, N-butoxymethyl -N- (Met Cymethyl) methacrylamide, N, N-di (pentoxymethyl) acrylamide, N-methoxymethyl-N- (pentoxymethyl) methacrylamide, N, N-dimethylaminopropylacrylamide, N, N-diethylaminopropylacrylamide, N , N-dimethylacrylamide, N, N-diethylacrylamide, monomers having primary to tertiary amide groups such as diacetone (meth) acrylamide;
(Meth) acrylic acid dimethylaminoethyl methyl chloride salt, trimethyl-3- (1- (meth) acrylamide-1,1-dimethylpropyl) ammonium chloride, trimethyl-3- (1- (meth) acrylamidopropyl) ammonium chloride, And monomers having a quaternary amino group such as trimethyl-3- (1- (meth) acrylamide-1,1-dimethylethyl) ammonium chloride;
Polyethylene glycol (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, propoxypolyethylene glycol (meth) acrylate, n-butoxypolyethylene glycol (meth) acrylate, n-pentoxypolyethylene glycol (meth) Acrylate, phenoxypolyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate, methoxypolypropylene glycol (meth) acrylate, ethoxypolypropylene glycol (meth) acrylate, propoxypolypropylene glycol (meth) acrylate, n-butoxypolypropylene glycol (meth) acrylate N-Pentoxypolypropylene Recall (meth) acrylate, phenoxypolypropylene glycol (meth) acrylate, polytetramethylene glycol (meth) acrylate, methoxypolytetramethylene glycol (meth) acrylate, phenoxytetraethylene glycol (meth) acrylate, hexaethylene glycol (meth) acrylate, And monomers having a polyether chain such as methoxyhexaethylene glycol (meth) acrylate.

  Among them, the other monomer is preferably a monomer having an alkyl group having 1 to 22 carbon atoms. By copolymerizing the monomers, it is possible to adjust the polarity and the like, to easily control the adsorption and desorption to the protein, and to improve the base material adhesion and stability with time of the vinyl polymer (A2).

<Betainization>
As described above, the polymer (A2) of the present invention is a reaction product of a vinyl polymer having a structure of at least any tertiary amino group represented by the general formula 6 or 7 in the side chain and a betaine agent. Specifically, a vinyl polymer obtained by polymerizing a monomer having a structure of at least any tertiary amino group represented by the general formula 6 or 7 and another monomer, a betaine agent, The reaction product of Betainization may be performed either before or after polymerization, and a betaine agent is added to a solution containing a betaine structure, and the temperature is in the range of 20 ° C to 100 ° C for 0.1 to 100 hours, preferably 1 to 50 hours. A betaine polymer can be obtained by reacting for a period of time. In general, the amount of the betaine agent used is 0.2 to 3 times the molar equivalent of the functional group capable of betainization, that is, the tertiary amino group, and further 0.5 to 2 times. More preferably, molar equivalents are used.
The betaine agent is synonymous with the betaine agent described in the item of the urethane-based polymer.

<Copolymerization composition of polymer (A2)>
The vinyl polymer (A2) is a monomer having at least one structure represented by (I) general formula 4 or 5 described above (provided that the bonding position with the main chain of the vinyl polymer in general formula 4 or 5 is In the case of a vinyl polymer obtained by polymerizing other monomers, the total number of monomers having a structure represented by formulas 4 to 5 is 1 in 100 mol% of the total monomers. It is preferable that it is -95 mol%, More preferably, it is 2-80 mol%.
The vinyl polymer (A2) is a reaction product of a vinyl polymer having at least any tertiary amino group structure represented by the following general formula (6) or (7) below in the side chain and a betaine agent. It is a polymer that is a product. Specifically, a monomer having a structure of at least any tertiary amino group represented by the following general formula 6 or 7 (provided that the bonding position with the main chain of the vinyl polymer in the general formula 6 or 7 is a monomer) In the case of a reaction product of a vinyl polymer obtained by polymerizing other monomers and a betaine agent, monomers having a structure represented by general formulas 6 to 7 and other monomers And in the total 100 mol% of the monomer (r), the monomer having a structure represented by the general formulas 6 to 7 is preferably 1 to 95 mol%, more preferably 2 to 80 mol%.

[Betaine structure content]
The betaine structure content of the vinyl polymer (A2) in the present invention is preferably 0.25 to 1.5 mmol / g, more preferably 0, from the viewpoint of ensuring cytotoxicity and wettability to the silicone substrate. 0.3 to 1.3 mmol / g, and more preferably 0.2 to 1.2 mmol / g. The betaine structure content can be calculated by an analytical method such as NMR or GC-MS.

[Mass average molecular weight (Mw)]
The mass average molecular weight of the vinyl polymer (A2) is preferably 2,000 or more, preferably 2,000 to 10,000,000, more preferably 5,000 or more, and still more preferably 10, 000 or more, and particularly preferably 100,000 or more. When the molecular weight is 2,000 or more, the cohesive force of the coating film can be imparted, peeling from the coated substrate can be suppressed, and a long-term cell adhesion preventing effect can be exhibited. Moreover, since it becomes an appropriate viscosity as it is below 10,000,000, since a coating suitability improves, it is preferable. More preferably, it is 500,000 or less.
As the mass average molecular weight of the vinyl polymer (A2), a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) is adopted. The measuring device and measurement conditions are basically based on the following condition 1 and are allowed to be set to condition 2 depending on the solubility of the sample. However, depending on the polymer species, an appropriate carrier (eluent) and a column suitable for it may be selected and used. For other matters, refer to JISK7252-1-4: 2008. In addition, it is assumed that the hardly soluble polymer compound is measured at a soluble concentration under the following conditions.
Moreover, when the molecular weight measurement of a vinyl type polymer (A2) is difficult, the mass average molecular weight of a betaine-ized precursor polymer can be made into the mass average molecular weight of a vinyl type polymer (A2). The mass average molecular weight of the betaened precursor polymer is a value measured in terms of standard polystyrene by gel permeation chromatography (GPC), and the measurement apparatus and measurement conditions are basically based on the following condition 3.

(Condition 1)
Column: TOSOHTSKgelSuperHZM-H,
TOSOHTSKgelSuperHZ4000,
TOSOHTSKgelSuperHZ2000
Carrier using tetrahydrofuran-connected column: Tetrahydrofuran Measurement temperature: 40 ° C
Carrier flow rate: 1.0 ml / min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection amount: 0.1 ml

(Condition 2)
Column: TosohtskegelSuperAWM-H Two carriers: 10 mM LiBr / N-methylpyrrolidone Measurement temperature: 40 ° C.
Carrier flow rate: 1.0 ml / min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection amount: 0.1 ml

(Condition 3)
Column: TOSOHTSKgelSuperAW4000,
TOSOHTSKgelSuperAW3000,
TOSOHTSKgelSuperAW2500
Use a column connected to
Carrier: N, N-dimethylformamide (1 L), triethylamine (3.04 g), mixed solution of LiBr (0.87 g) Measurement temperature: 40 ° C.
Carrier flow rate: 0.6ml / min

<Crosslinking agent>
The cell culture equipment treating agent of the present invention can further contain a crosslinking agent. By including a cross-linking agent, particularly when the above-described vinyl polymer (A2) has a cross-linkable group, it is possible to improve the durability (water resistance) by forming a cross-link in the coating film.
The crosslinking agent that can be used in the present invention reacts with at least one crosslinking group selected from the group consisting of a carboxyl group and a hydroxyl group in the monomer (r) for forming the vinyl polymer (A2). For example, examples of the crosslinking agent for the vinyl polymer (A2) having a carboxyl group introduced include isocyanate compounds, aziridine compounds, carbodiimide compounds, and amino compounds. Examples of the crosslinking agent for the vinyl polymer (A2) having a hydroxyl group introduced include carbodiimide compounds, isocyanate compounds, amino compounds, and alkoxylane compounds. These cross-linking agents can be used for the purpose of increasing the elastic modulus and resistance of the coating film, or for adjusting the adhesion to the substrate.
Below, a carbodiimide compound is demonstrated as an example of a preferable crosslinking agent.

[Carbodiimide group-containing compound (carbodiimide compound)]
Examples of the carbodiimide group-containing compound include dicarbodiimides such as N, N′-diisopropylcarbodiimide, N, N′-dicyclohexylcarbodiimide, N, N′-di-2,6-diisopropylphenylcarbodiimide, and poly (1,6-hexa). Methylene carbodiimide), poly (4,4′-methylenebiscyclohexylcarbodiimide), poly (1,3-cyclohexylenecarbodiimide), poly (1,4-cyclohexylenecarbodiimide), poly (4,4′-dicyclohexylmethanecarbodiimide) , Poly (4,4′-diphenylmethanecarbodiimide), poly (3,3′-dimethyl-4,4′-diphenylmethanecarbodiimide), poly (naphthalenecarbodiimide), poly (p-phenylenecarbodiimide), poly (m-phenylenecarbohydrate) Diimide), poly (tolylcarbodiimide), poly (diisopropylcarbodiimide), poly (methyl-diisopropylphenylenecarbodiimide), poly (1,3,5-triisopropylbenzene) polycarbodiimide, poly (1,3,5-triisopropylbenzene) And polycarbodiimides such as polycarbodiimide, poly (1,5-diisopropylbenzene) polycarbodiimide, poly (triethylphenylenecarbodiimide), and poly (triisopropylphenylenecarbodiimide).
Examples of commercially available carbodiimide group-containing compounds include “Carbodilite” (registered trademark) manufactured by Nisshinbo Holdings, Inc., and “Stavaxol (registered trademark)” manufactured by Rhein Chemie.

  In the present invention, the crosslinking agent may be used alone or in combination. The amount of the crosslinking agent used may be determined in consideration of the type and mole number of the functional group contained in the vinyl polymer (A2), and is not particularly limited, but is usually vinyl polymer (A2). It is used in the range of 0.1 to 100 parts by mass with respect to 100 parts by mass. By blending in a range smaller than the number of moles of the functional group contained in the vinyl polymer (A2), the concern that the unreacted crosslinking agent is liberated can be eliminated. If it is this range, the especially outstanding performance will be expressed in each effect of the target cell culture equipment processing agent.

<Preparation of cell culture equipment treatment agent>
The cell culture equipment treating agent of the present invention is the urethane polymer (A1) or the vinyl polymer (A2) (hereinafter referred to as polymer (A1) or (A2)) in 100% by mass of the cell culture equipment treating agent. The content is preferably 50% by mass or less, and more preferably 0.5 to 10% by mass. By setting the content of the polymer (A1) or (A2) to 0.5% by mass or more, the effect of inhibiting cell adhesion by the betaine group can be exhibited. Moreover, the cell culture equipment treating agent of the present invention may contain components other than the polymer (A1) or (A2).

<Solvent>
The cell culture equipment treating agent of the present invention may contain a solvent as a component other than the polymer (A1) or (A2), or may contain two or more kinds in combination. The solvent can be appropriately selected from conventionally known solvents in consideration of the solubility of the polymer (A1) or (A2) depending on the amount of betaine, film forming conditions, and the like.
For example, when the amount of betaine in the polymer (A1) or (A2) is large, water, alcohols such as methanol and ethanol, ketones such as acetone and ethyl methyl ketone, ethers such as tetrahydrofuran and diethyl ether, methyl acetate, Esters such as ethyl acetate, dimethyl sulfoxide, acetonitrile, organic acids such as formic acid and acetic acid, and organic bases such as N, N-dimethylformamide can be selected. On the other hand, when the amount of betaine in the polymer (A1) or (A2) is small, ketones such as acetone and ethyl methyl ketone, ethers such as tetrahydrofuran and diethyl ether, esters such as methyl acetate and ethyl acetate, dimethyl sulfoxide In addition to acetonitrile, halogen solvents such as dichloromethane and trichloromethane can be selected.

<Other additives>
The cell culture equipment treatment agent of the present invention may contain various additives as long as the effects of the present invention are not impaired. A surfactant, an isotonic agent, a chelating agent, a pH adjuster, and a buffering agent. , Thickeners, stabilizers, proteolytic enzymes, pharmacologically active ingredients, physiologically active ingredients, various additives described in the Pharmaceutical Additives Dictionary, etc. may be included within a range not impairing the effects of the present invention. . These may contain 1 type independently and may contain 2 or more types.

  For example, a pH adjusting agent may be contained in order to adjust the ion balance. Examples of the pH adjuster include acidic or basic compounds. Examples of acidic pH adjusters include carboxylic acids, inorganic acids and sulfonic acids, and examples of basic pH adjusters include nitrogen-containing compounds other than hydroxides, alkoxides, primary and secondary amines. , Basic carbonates, and basic phosphates, but are not limited to these. The pH value of the cell culture equipment treatment agent is about 4.0 to 9.0, preferably about 6.0 to 8.0, and is preferably adjusted to be around 7.0.

  Examples of isotonic agents include sugars, polyalcohols (such as mannitol and sorbitol), and sodium chloride. As thickeners and stabilizers, synthetic organic polymer compounds such as polyvinyl alcohol, poly-N-vinylpyrrolidone, polyethylene glycol, polypropylene glycol, polyacrylamide, and cellulose derivatives such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc. , Starch derivatives such as sodium carboxymethyl starch and hydroxyethyl starch, chondroitin sulfate, hyaluronate and the like.

<Cell culture equipment>
The cell culture device of the present invention has a cell adhesion preventive film (also referred to as a coating film) containing the polymer (A1) or (A2) on the substrate, and the polymer (A1) or It can be produced by including a step of forming a cell adhesion-preventing film containing (A2), specifically, a step of coating a cell culture equipment treating agent containing polymer (A1) or (A2). .

  The method for forming the cell adhesion preventing film, that is, the step of coating the cell culture device treating agent containing the polymer (A1) or (A2) on the substrate can be performed by a known method. For example, a coating film can be formed by coating the cell culture equipment treating agent by a known method and then performing a heat treatment or the like. In addition, it can also harden | cure using a crosslinking agent.

  The coating method can be appropriately selected depending on the substrate, and specific examples include spray coating method, dip coating method, flow coating method, ink jet method, brush coating, sponge coating and the like. Various printing methods such as gravure printing, offset printing, and silk screen printing may be used. Although the heat processing temperature after coating can be suitably selected according to the boiling point of the solvent to contain and the crosslinking temperature of a crosslinking agent, it is desirable that it is 40-180 degreeC industrially. Furthermore, when this composition contains a crosslinking agent, it is preferable to provide the process for accelerating | stimulating a crosslinking reaction. The crosslinking conditions are generally 40 to 150 ° C. and 6 to 24 hours, but are not limited thereto.

<Base material>
The substrate of the present invention is not particularly limited as long as it can be used for cell culture equipment. Specifically, (meth) acrylic resin, styrene resin, polycarbonate resin, polyolefin resin, polystyrene, polyethylene, polyvinyl chloride, polypropylene, polyester, polyvinyl acetate, vinyl acetate copolymer, styrene methyl methacrylate Acrylate copolymer, acrylonitrile-styrene copolymer, acrylonitrile-butadiene-styrene copolymer, nylon, polymethylpentene, silicone resin, amino resin, polysulfone, polyethersulfone, polyetherimide, fluororesin, and In addition to one or more kinds of resins selected from the group consisting of polyimide resin materials, glass substrates and the like can be mentioned.

<Silicone substrate>
The silicone substrate is not particularly limited as long as it is a substrate containing a silicone resin.
Examples of the silicone-based resin include those having a main chain of polyorganosiloxane such as polydimethylsiloxane, polyphenylmethylsiloxane, and polydiphenylsiloxane, and polydimethylsiloxane is preferable. Specific examples of such a silicone substrate include a silicone-containing medical device, a silicone substrate having a microchannel, and preferably a silicone-containing microchannel device.

The medical device refers to a medical structure used in a living body, and such a structure is used by being implanted in the body, and is used in the body (without being implanted). It is divided roughly into. In addition, the size and length of the medical device are not particularly limited, and include a device having a fine circuit and a device that detects a small amount of sample.
As a structure to be used by being implanted in the body, for example, a function assisting device for supplementing the function of a living body having a disease such as a cardiac pacemaker; a device for detecting an abnormality of the living body such as an implantable biochip; Medical devices such as implants, bone anchors, sutures, and artificial blood vessels can be mentioned. Examples of structures used in the body (not implanted) include catheters and gastrocameras.

  Examples of the microchannel device include a microreaction device (specifically, a microreactor and a microplant), a microanalysis device such as an integrated nucleic acid analysis device, a microelectrophoresis device, and a microchromatography device; Micro devices for sample preparation such as liquid chromatography; physicochemical processing devices used for extraction, membrane separation, dialysis, etc .; environmental analysis chip, clinical analysis chip, gene analysis chip (DNA chip), protein analysis chip (proteome chip) And microchannel chips such as sugar chain chips, chromatographic chips, cell analysis chips, and pharmaceutical screening chips. Among these, a microchannel chip is preferable.

The microchannel is a portion through which a small amount of sample (preferably a liquid sample) flows, and the channel width and depth are not particularly limited, but both are usually about 0.1 μm to 1 mm, Preferably they are 10 micrometers-800 micrometers. Note that the channel width and depth of the microchannel may be the same over the entire length of the channel, or may be partially different in size and shape.
The silicone substrate may be subjected to plasma treatment, UV ozone treatment, internal infiltrant treatment, and the like. Since the surface of the silicone base material subjected to these treatments is made hydrophilic, it may be difficult to adsorb the polymer, but the polymer (A1) or (A2) used in the present invention is such a plasma treatment. It is also adsorbed to a silicone base material that has been subjected to the treatment, etc., and imparts excellent hydrophilicity and biological sample adsorption suppression effect.

<Medical device>
The medical device is not particularly limited as long as it is a medical device, and specific examples include, for example, a blood bag, a urine collection bag, a blood transfusion set, a suture, a drain tube, various catheters, a blood access, a blood circuit, an artificial blood vessel, Artificial kidney, heart-lung machine, artificial valve, plasma exchange membrane, various adsorbents, CAPD, IABP, pacemaker, artificial joint, artificial bone head, dental material, various shunts.
The material and shape of the medical device are not particularly limited. Examples of the material include polyolefin, modified polyolefin, polyether, polyurethane, polyamide, polyimide, polyethylene terephthalate, polyester, polytetrafluoroethylene, polyvinyl chloride, and copolymers thereof. And various polymer materials such as metals, ceramics, carbon, and composite materials thereof. Further, the shape is not particularly limited, and any shape such as smooth or porous may be used.

  The following examples further illustrate the present invention, but the following examples do not limit the scope of the present invention in any way. In the examples, “parts” and “%” represent “parts by mass” and “mass%”, respectively.

<Measurement method of mass average molecular weight (Mw)>
As the mass average molecular weight of the polymer (A1) or (A2), a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) was adopted. The measurement apparatus and measurement conditions were based on the following condition 1 and were set to condition 2 depending on the solubility of the sample. However, depending on the polymer type, an appropriate carrier (eluent) and a column suitable for the carrier were selected as appropriate. About other matters, it was based on JISK7252-1-4: 2008. In addition, about the sparingly soluble high molecular compound, it measured by the density | concentration which can be melt | dissolved under the following conditions.
Moreover, when it was difficult to measure the molecular weight of the polymer (A1) or (A2), the mass average molecular weight of the betaine precursor polymer was taken as the mass average molecular weight of the polymer (A1) or (A2). As the mass average molecular weight of the betaine precursor polymer, a value measured in terms of standard polystyrene by gel permeation chromatography (GPC) was adopted, and the measurement apparatus and measurement conditions were based on the following condition 3.
(Condition 1)
Column: TOSOHTSKgelSuperHZM-H,
A combination of TOSHOTSKgelSuperHZ4000 and TOSHOTSKgelSuperHZ2000.
Carrier: Tetrahydrofuran Measurement temperature: 40 ° C
Carrier flow rate: 1.0 mL / min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection amount: 0.1 mL
(Condition 2)
Column: A column in which two TOSOHTSKgelSuperAWM-H are linked.
Carrier: 10 mM LiBr / N-methylpyrrolidone Measurement temperature: 40 ° C.
Carrier flow rate: 1.0 mL / min
Sample concentration: 0.1% by mass
Detector: RI (refractive index) detector Injection amount: 0.1 mL
(Condition 3)
Column: TOSOHTSKgelSuperAW4000,
A combination of TOSHOTSKgelSuperAW3000 and TOSHOTSKGgelSuperAW2500.
Carrier: N, N-dimethylformamide (1 L), triethylamine (3.04 g), mixed solution of LiBr (0.87 g) Measurement temperature: 40 ° C.
Carrier flow rate: 0.6 mL / min

<Method for measuring hydroxyl value>
The hydroxyl value was measured by the method described in JISK-1557.

<Manufacture of urethane-based polymer (A1)>
[Preparation of Urethane Polymer (A1-1p)]
A 4-necked flask equipped with a stirrer, a reflux condenser, a nitrogen inlet pipe, an inlet pipe, and a thermometer, 10 parts of N-methyldiethanolamine as a monomer having a tertiary amino group, and Sanix PP4000 (hydroxyl value: 26.8 KOH mg / g (manufactured by Sanyo Chemical Co., Ltd.) was charged with 77 parts, and 19 parts of hexamethylene diisocyanate was added as a polyisocyanate component. The mixture was heated to 60 ° C. with stirring in a nitrogen stream and dissolved uniformly. Subsequently, 0.1 part of dibutyltin dilaurate was added thereto as a catalyst and reacted at 110 ° C. for 3 hours.
Thereafter, the temperature was lowered to 40 ° C., and 1 part of isophoronediamine as a polyamine component was dropped over 2 hours to carry out a chain extension reaction. Furthermore, 0.1 part of hexylamine as a terminal terminator was added and reacted at 40 ° C. for 30 minutes to obtain a solution of a betaine precursor polymer, that is, a polymer having a tertiary amino group.
Next, 11 parts of 1,4-butane sultone (equal molar amount with the used N-methyldiethanolamine) is added as a betaine agent to the polymer solution having the tertiary amino group, and reacted at 70 ° C. for 10 hours. Thus, betaine formation of the amino group was performed. After confirming that the amine oxide conversion rate exceeded 99%, the product was cooled and taken out, and then the solvent was completely volatilized in an oven to obtain a urethane bond and urea bond polymer (A1-1p). The weight average molecular weight of the obtained polymer was 201,500.

[Preparation of urethane polymer (A1-2p to 7p)]
Polymers (A1-2p to 7p) were synthesized by the same method as (A1-1p) with the composition shown in Table 1.

Abbreviations in Table 1 are shown below.
<Monomer having a tertiary amino group (polyol having a tertiary amino group)>
MDEA: N-methyldiethanolamine EDEA: N-ethyldiethanolamine DMAP: 3-dimethylaminopropane-1,2-diol <Polyol>
PP4000: Sannix PP4000 (polyoxypropylene glycol, hydroxyl value: 26.8 KOHmg / g, manufactured by Sanyo Chemical Industries, Ltd.)
P4010: Kuraray polyol P4010 (aliphatic polyester polyol, hydroxyl value 28 KOHmg / g, manufactured by Kuraray Co., Ltd.)
GI-3000: hydroxylated hydrogenated polybutadiene at both ends (hydroxyl value: 27.7 KOHmg / g, manufactured by Nippon Soda Co., Ltd.)
<Polyisocyanate component>
HDI: Hexamethylene diisocyanate IPDI: Isophorone diisocyanate <Polyamine component>
IPDA: isophoronediamine HA: hexylamine B1: 1,4-butane sultone B2: sodium 4-probutanesulfonate B3: β-propiolactone B4: sodium 2-chloroacetate

<Production of vinyl polymer (A2)>
The 1-vinyl-3- (3-sulfopropyl) imidazolium inner salt and 2-vinyl-1- (3-sulfopropyl) pyridinium inner salt used for the synthesis of the vinyl polymer are described in paragraphs of JP-A No. 08-218065. Synthesized with reference to 0018.

[Vinyl polymer (A2-1p)]
A reaction vessel equipped with a gas introduction tube, a thermometer, a condenser, and a stirrer was charged with 184 parts of isopropyl alcohol and replaced with nitrogen gas, and then heated to 75 ° C. and stirred. Next, 0.1 part of 2,2′-azobis (2,4-diethylvaleronitrile), 50 parts of 1-vinyl-3- (3-sulfopropyl) imidazolium inner salt, 10 parts of methyl methacrylate, A solution comprising 30 parts of dicyclopentenyloxyethyl acrylate, 10 parts of 2-methoxyethyl acrylate, 75 parts of methanol, 4 parts of methyl ethyl ketone, and 2 parts of water is prepared and added dropwise from a dropping funnel over 2 hours to conduct a polymerization reaction. went. After completion of the dropwise addition, the reaction was further continued at 75 ° C. for 3 hours. After confirming that the conversion rate exceeded 98% by solid content measurement, the reaction was stopped by cooling. Thereafter, isopropyl alcohol, methanol, methyl ethyl ketone, and water were removed with a diaphragm pump to obtain a vinyl polymer (A2-1p). The weight average molecular weight of the vinyl polymer (A2-1p) was 213,000.

[Vinyl polymer (A2-2p to 5p)]
With the composition shown in Table 2, vinyl polymers (A2-2p to 5p) were synthesized in the same manner as the vinyl polymer (A2-1p).

[Vinyl polymer (A2-6p)]
A reaction vessel equipped with a gas introduction tube, a thermometer, a condenser, and a stirrer was charged with 98.0 parts of 1-butanol and replaced with nitrogen gas. The reaction vessel was heated to 110 ° C. to give 50 parts of butyl acrylate, 30 parts of dicyclopentenyloxyethyl acrylate, 10 parts of methyl methacrylate, and 2,2′-azobis (2,4-diethylvaleronitrile). 0.1 part of the mixture was added dropwise over 2 hours to carry out the polymerization reaction. After completion of the dropping, the reaction was further carried out at 110 ° C. for 3 hours, and after confirming that the conversion rate exceeded 98% by solid content measurement, the reaction was stopped by cooling to room temperature. Thereafter, 1-butanol was removed with a diaphragm pump to obtain a vinyl polymer for comparison. Its mass average molecular weight was 239,000.

[Vinyl polymer (A2-7p)]
According to Synthesis Example 8 of International Publication No. 2017/022815, a vinyl polymer (A2-7p) derived from 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as MPC) was obtained. Its mass molecular weight was 151,000.

[Vinyl polymer (A2-8p)]
A reaction vessel equipped with a gas introduction tube, a thermometer, a condenser, and a stirrer was charged with 49.4 parts of ethyl acetate and 50 parts of ethanol, replaced with nitrogen gas, and then heated to 75 ° C. and stirred. Next, 0.13 part of 2,2′-azobis (2,4-diethylvaleronitrile), 0.4 part of methyl ethyl ketone, 50 parts of 1-vinylimidazole, 20 parts of methyl methacrylate, dicyclopentenyloxyethyl A solution consisting of 30 parts of acrylate was prepared and dropped from the dropping funnel over 2 hours to conduct a polymerization reaction. After completion of the dropwise addition, the mixture was further reacted at 75 ° C. for 3 hours. After confirming that the conversion rate exceeded 98% by solid content measurement, 109 parts of 1,4-butane sultone was added, and stirring was further continued for 20 hours.
Subsequently, the mixture was cooled and taken out, and ethyl acetate, ethanol, and methyl ethyl ketone were completely volatilized with a diaphragm pump. The dried resin was thoroughly washed with methyl ethyl ketone to remove by-products and remaining raw materials. The obtained vinyl polymer (A2-8p) had a mass average molecular weight of 211,000.

[Vinyl polymer (A2-9p to 11p)]
Vinyl-based polymers (A2-9p to 11p) were synthesized by the same method as (A2-8p) with the composition shown in Table 3.

Abbreviations in Tables 2 and 3 are shown below.
VSPI: 1-vinyl-3- (3-sulfopropyl) imidazolium inner salt VSPP: 2-vinyl-1- (3-sulfopropyl) pyridinium inner salt VI: 1-vinylimidazole VP: 2-vinylpyridine MMA: methacryl Acid methyl DCPA: dicyclopentenyloxyethyl acrylate BA: butyl acrylate St: styrene MEA: 2-methoxyethyl acrylate AA: acrylic acid HEA :: 2-hydroxyethyl acrylate V65: 2,2′-azobis (2, 4-diethylvaleronitrile)

[Manufacture of cell culture equipment treatment agents]
[Example 1]
(Cell culture equipment treatment agent (AP-1)
The obtained polymer (A1-1p) was diluted with ethanol to prepare a 10% solution to obtain a cell culture equipment treating agent (AP-1).

[Examples 2 to 12, Comparative Examples 1 to 3]
(Cell culture equipment treatment agent (AP-2 to 12, 16 to 18)
The cell culture equipment treatment agent (AP-2 to 12, 16 to 18) was prepared in the same manner as the cell culture equipment treatment agent (AP-1) except that the polymer was changed to the polymer shown in Table 4. .

[Examples 13 to 15]
(Cell culture equipment treatment agent (AP-13-15))
10.0 parts of the obtained polymer (A2-3p) and 5.0 parts of a carbodiimide group-containing compound (manufactured by Nisshinbo Holdings Inc .; Carbodilite V-02) as a crosslinking agent were diluted with ethanol to obtain a 10% solution. The cell culture equipment treating agent (AP-13) was prepared.
A cell culture equipment treatment agent (AP-14 to 15) was prepared in the same manner as the cell culture equipment treatment agent (AP-13) except that the polymer was changed to the polymer shown in Table 4.

Abbreviations in Table 4 are shown below.
V02: Carbodiimide group-containing compound (Nisshinbo Holdings, Inc .; Carbodilite V-02)

<Manufacture of cell culture equipment>
[Examples 16 to 30, Comparative Examples 4 to 6]
(Cell culture equipment (D-1 to 18))
The obtained cell culture equipment treatment agent (AP-1-18) was poured into a PDMS (polydimethylsiloxane) standard chip (fluidware technologies) channel having a channel having a width of 200 μm and a height of 50 μm, and 50 After centrifuging for 4 hours in a heated state at 0 ° C., the cells were washed three times with pure water and once with PBS (manufactured by Hayashi Pure Chemical Industries, Ltd .; phosphate buffered saline) to remove unadsorbed components, and the cells Culture equipment (D-1 to 18) was obtained.

[Examples 31-45, Comparative Examples 7-9]
(Cell culture equipment (S-1-18))
On a silicone rubber seed (thickness: 5 mm, manufactured by Togawa Rubber), a cell culture equipment treatment agent (AP-1 to 18) is spin-coated so as to have a film thickness of 1.0 μm, and heat-treated at 90 ° C. for 2 minutes. Cell culture equipment (S-1 to 18) coated on a glass with a cell culture equipment treatment agent (AP-1 to 18) was obtained.

[Examples 46 to 60, Comparative Examples 10 to 12]
(Cell culture equipment (U-1-18))
About 0.5 ml of cell culture equipment treatment agent (AP-1 to 18) was injected into each well into a U-shaped 96-well plate. After sucking and discharging this, it was dried at 50 ° C. for 3 hours to produce cell culture equipment (U-1-18) in which the well plate was coated with the cell culture equipment treatment agent (AP-1-18). did.

[Evaluation of cell culture equipment treatment]
About the cell culture equipment (D-1-18), (S-1-18), and (U-1-18) using the obtained cell culture equipment processing agent (AP-1-18), the following evaluation was carried out. Carried out. The results are shown in Tables 5-7.

[Evaluation Item 1. <Blood feeding test>]
In the obtained cell culture equipment (D-1 to 18), a blood sample was fed at a constant pressure for 12 minutes at an inlet speed of 2 μL / min. The amount of blood sample that flowed out, the amount of blood sample that flowed out from the channel outlet in 1 minute after 5 minutes from the start of liquid feeding, and the channel outlet from 1 minute after 11 minutes from the start of liquid feeding The amount of blood sample that flowed out was measured. The larger the amount of blood sample that has flowed out, the better as it is constant without decreasing over time.

[Evaluation Item 2. <Transparency evaluation>
About the obtained cell culture equipment (D-1-18), the light transmittance in 450 nm was measured using the ultraviolet spectrophotometer. The higher the light transmittance, the better the transparency. Evaluation was made according to the following criteria.
Evaluation criteria:
A: The light transmittance is 95% or more. Very good.
A: The light transmittance is 90% or more and less than 95%. Good.
Δ: Light transmittance is 85% or more and less than 90%. Available.
X: Light transmittance is 85% or less. Bad.

[Evaluation Item 3. <Base material adhesion evaluation>]
<Adhesion>
About the obtained (S-1-18), the base-material adhesiveness of the cell culture equipment processing agent was evaluated by the cross-cut tape method according to JISK5400 (1990 edition).
(Evaluation criteria)
A: 100/100 (all adhesion and substrate adhesion are very good.
A: 99/100 to 80/100 (good substrate adhesion)
Δ: 79/100 to 50/100 (partially peeled but usable)
X: 49 / 100-0 / 100 (partially peeled-all peeled, defective)

[Evaluation Item 4. <Antibody protein adsorptivity>]
Cell culture equipment (S-1-18) was cut into 11 mm squares into a flat-bottomed 24-well plate, and 1 ml of HPR-IgG solution diluted 10,000 times with phosphate buffered saline (PBS) was added and immersed. I let you. After 1 hour incubation at room temperature, each well was washed 4 times with PBS-T (0.1% Tween 20). After 1 ml of the staining solution was dispensed to each well and incubated at room temperature for 10 minutes, 1 ml of the reaction stop solution was dispensed to each well, and the absorbance Aλ of 450 nm (subwavelength 650 nm) was measured using the MITHRAS2 LD943-M2M microplate. Measurement was performed using a reader. The smaller the absorbance Aλ, the lower the antibody protein adsorptivity and the better. Evaluation was made according to the following criteria.
Staining solution: TMBZ solution (3,3 ', 5,5'-tetramethylbenzidine)
Reaction stop solution: TAKARA BIO INC. WASH and Stop Solution For ELISA With Solution for ELISA Acid Without Sulfur Acid
HPR-IgG: Enzyme, Antibody (HORSERADIH PEROXIDASE IMMUNOGLLOBULING)
PBS-T: 0.1% Tween 20 added to PBS (Evaluation criteria)
A: Aλ ≦ 0.2 (very good)
○: 0.2 <Aλ ≦ 0.6 (good)
Δ: 0.6 <Aλ ≦ 0.8 (available)
×: 0.8 <Aλ (defect)

[Evaluation Item 5. <Spheroid formation test>
Cell culture equipment (U-1 to 18) was sterilized with ethylene oxide gas, then Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) was used as the culture medium, and a mouse fibroblast cell line ( NIH / 3T3 cells) were seeded 1 × 10 ⁴ cells per well, and cultured until day 5 with 5% _CO 2/37 ℃ incubator, photography cell culture conditions after 5 days with a transmission type optical microscope 40 times And evaluated by observing cell morphology.
(Evaluation criteria)
○: One spheroid is formed Δ: A plurality of spheroids are formed ×: No spheroid is formed

As shown in Tables 5 to 7, the cell culture equipment using the cell culture equipment treatment agent of the present invention has suppressed antibody protein adsorptivity and showed excellent blood fluidity. Moreover, what used the urethane type polymer which has any structure represented by General formula 1-3 was especially excellent in transparency and base-material adhesiveness.
On the other hand, when resin (AP-16) and (AP-17) which do not have a betaine structure were included, it resulted in spheroid formation property, protein adsorption | suction suppression, and blood liquid feeding property being greatly inferior.
As described above, the cell culture equipment using the cell culture equipment treatment agent of the present invention has high spheroid-forming properties, excellent protein, cell adhesion prevention effect, and excellent blood fluidity and transparency. Therefore, it was shown to be useful as a medical device.

Claims (10)

The cell culture equipment processing agent containing the urethane type polymer (A1) which has at least any one structure shown by the following general formulas 1-3.
General formula 1

General formula 2

General formula 3

(In general formulas 1 to 3,
R ¹ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
R ² , R ⁴ and R ⁷ are each independently an alkylene group having 1 to 6 carbon atoms,
R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ are each independently an alkyl group, aryl group, aralkyl group, or pyridyl group,
Y is -COO ^- or -SO ₃ ^-,
X is an oxygen atom or -NH-,
* Represents the bonding position with the main chain of the urethane-based polymer. ) A cell culture equipment treating agent comprising a vinyl polymer (A2) having a side chain having at least one structure represented by the following general formula 4 or 5.
Formula 4

Formula 5

(In general formulas 4 and 5,
Y is -COO ^- or -SO ₃ ^-,
4 of R ^{10 to} R ¹⁴ are a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
One of R ^{10 to} R ¹⁴ is a bonding position with the main chain of the vinyl polymer,
R ¹⁵ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
R ¹⁶ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ¹⁷ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms,
* Represents a bonding position with the main chain of the vinyl polymer. )   The cell culture equipment treatment agent according to claim 1, wherein the urethane-based polymer (A1) contains at least one of the structures represented by the general formulas 1 to 3 in a total amount of 0.25 to 1.5 mmol / g.   The cell culture equipment treating agent according to claim 2, wherein the vinyl polymer (A2) has at least one crosslinkable group selected from the group consisting of a carboxyl group and a hydroxyl group.   Furthermore, the cell culture equipment processing agent of any one of Claims 1-4 containing a crosslinking agent.   The cell culture equipment which has a cell adhesion prevention film formed from the cell culture equipment treatment agent of any one of Claims 1-5 on a base material.   The cell culture device according to claim 6, wherein the substrate is a silicone substrate.   The cell culture device according to claim 6 or 7, wherein the substrate is a silicone substrate having a microchannel.   The manufacturing method of a cell culture equipment including the process of forming the cell adhesion prevention film formed from the cell culture equipment treatment agent of any one of Claims 1-5 on a base material.   A medical device comprising the cell culture device according to claim 6.