JP6488163B2 - Cell culture substrate - Google Patents

Description

  The present invention relates to a cell culture substrate, a cell culture container provided with the substrate, a method of culturing cells using the substrate, and forming a three-dimensional tissue from cells using the substrate. On how to do.

  Cells that form organs such as the liver, pancreas, skin, and blood vessels are three-dimensionally forming a network and expressing their functions in vivo.

  Therefore, in research on regenerative medicine that regenerates the functions of these organs, when cells of these organs are cultured, a culture in which cells can form a three-dimensional network (that is, three-dimensional culture) is required. . Spheroids are the smallest tissue in which cells form a three-dimensional network. However, when cells are cultured on the surface of a general resin-made cell culture substrate, the cells spread in a plane and grow, and a three-dimensional network is not formed.

Various materials have been developed as base materials for performing three-dimensional culture.
For example, Patent Document 1 discloses a cell culture sheet that includes a substrate formed from a thermoplastic organic polymer and a columnar microprojection group extending from the substrate, and in which cells are attached to the projections of the columnar microprojection group and cultured. Has been. Patent Document 2 discloses a cell content structure having a concavo-convex structure functioning as a cell adhesion surface, in which a plurality of microcells having a polygonal shape in the planar direction and a minimum inner diameter of 3 μm or less are continuously formed. Has been.

  In Non-Patent Document 1, focusing on the fact that fluorine-containing polyimide has excellent biocompatibility, cell culture was performed on the surface of 6FDA-6FAP, which is one type of fluorine-containing polyimide, and the surface was flat. On the 6FDA-6FAP membrane, cells are two-dimensionally proliferated and spheroids are not formed, whereas spheroids are formed when fine irregularities are formed on the surface by rubbing treatment. Here, 6FDA-6FAP means 2,2′-bis (3,4-dicarboxyphenyl) hexafluoropropane dianhydride (6FDA) which is an acid dianhydride and 2,2′-bis which is a diamine compound. It is a polyimide obtained by polymerizing (4-aminophenyl) hexafluoropropane (6FAP). In Patent Document 3, the authors of Non-Patent Document 1 cultivated vascular endothelial cells two-dimensionally on a flat 6FDA-6FAP membrane that has not been rubbed, and then transferred to a gel to form vascular tissue. Disclosed is a method of culturing liver cancer cells three-dimensionally on a 6FDA-6FAP film having a rubbed uneven surface to form spheroids and combining the two.

  Patent Documents 4 to 6 disclose a film and a film obtained from a fluorine-containing polyimide for forming an uneven structure and a resin composition containing the fluorine-containing polyimide.

  Patent Document 7 discloses a technique for forming a cell culture container from a gas-permeable plastic material. Patent Document 7 aims to provide a cell culture vessel that does not require an oxygen supply device.

Japanese Patent No. 4897192 Japanese Patent No. 4159103 JP 2009-213716 A JP 2014-83783 A JP 2014-210404 A JP 2015-17232 A Japanese Patent No. 3761676

N. Matsumoto et al. , Polymers for Advanced Technologies, 19, 1002 (2008)

  Each of the cell culture substrates for three-dimensional culture disclosed in Patent Documents 1 and 2 enables three-dimensional culture by forming a fine uneven structure on the surface of the base material. Such a base material has a problem that it is not easy to create fine irregularities and the processing cost is high. When cell culture is performed on the surface of a substrate having fine irregularities, bubbles are likely to remain on the substrate surface when the liquid medium is applied to the substrate surface. There was also a problem that foam treatment was necessary.

  Non-patent document 1 and patent document 3, disclosed in US Pat. Although possible, it has the same problems as in Patent Documents 1 and 2, and there is still room for improvement.

  Then, an object of this invention is to provide the base material for cell cultures provided with the fluorine-containing polymer in which a three-dimensional tissue culture is possible on the surface.

  On the other hand, the container of Patent Document 7 is not intended to form a three-dimensional structure, and it has not been studied at all whether the three-dimensional structure can be formed using the container of Patent Document 7.

  Accordingly, the present invention also aims to provide a cell culture substrate capable of three-dimensional tissue culture, a cell culture container provided with the substrate, and a cell culture method using the substrate. .

  The inventors of the present invention provide a three-dimensional tissue such as a spheroid or a three-dimensional cell aggregate on a substrate having a fluorine-containing polymer having a fluorine-containing aromatic ring and an ether bond in the main chain on the surface. We have found a surprising finding that can be formed.

Accordingly, in a first aspect, the present invention provides:
At least a part of the surface is constituted by a resin composition containing a fluorine-containing polymer,
The fluorine-containing polymer is a polymer having a fluorine-containing aromatic ring and having an ether bond in the main chain.

  The cell culture substrate having the above-described features enables cells to form a three-dimensional tissue on the surface. In addition, the base material of the present invention is easy to manufacture because it is not necessary to give a three-dimensional structure to the surface serving as a scaffold for cells.

In a preferred embodiment of the cell culture substrate, the oxygen gas permeability is 219 cm ³ (STP) / (m ² · 24 h · atm) or more.

In a preferred embodiment of the cell culture substrate, the resin composition has an oxygen gas permeability coefficient of 0.10 × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or more.

  According to the above-described embodiment, a large amount of oxygen is easily supplied to cells, and cell growth and formation of three-dimensional tissues such as spheroids and three-dimensional cell aggregates are likely to proceed.

Moreover, in suitable embodiment of the said base material for cell cultures, the fluorine content in the said resin composition is 1-60 mass%.
According to this embodiment, a three-dimensional structure on the substrate surface is easily formed, which is preferable.

〔式中、Ｒ^４２は以下の構造：

のいずれかである。〕
で表される繰り返し単位を含む含フッ素アリールエーテルケトンポリマーである。
この実施形態によれば、透明で適度に細胞が付着した３次元的な組織が形成する足場となる。また、細胞により多くの酸素が供給され易く、細胞の生育と三次元的な組織の形成が進み易い。 In a preferred embodiment of the cell culture substrate, the fluorine-containing polymer is represented by the following formula (II-1):

[Wherein R ⁴² has the following structure:

One of them. ]
It is a fluorine-containing aryl ether ketone polymer containing the repeating unit represented by these.
According to this embodiment, the scaffold is formed by a transparent and moderately attached three-dimensional tissue. In addition, more oxygen is easily supplied to the cells, and cell growth and three-dimensional tissue formation are likely to proceed.

  In addition, the present inventors used a base material containing a fluorine-containing polymer on the surface, for example, when the base material is placed on the bottom of a single or multi-type plate or petri dish, and more preferably, oxygen gas A substrate containing a highly permeable resin composition is used, and a cell and a medium are brought into contact with the surface of the substrate containing the fluorine-containing polymer. In addition to the upper surface of the medium, the other surface of the substrate contains oxygen such as air. When cell culture was carried out in contact with gas, the inventors discovered the surprising finding that cells can form three-dimensional tissues such as spheroids and three-dimensional cell aggregates, which led to the completion of the present invention. .

Thus, in a second aspect, the present invention provides
The present invention relates to a cell culture container, wherein at least a part of the container is composed of the cell culture substrate.

  When the container for cell culture of the present invention is used, since it has a culture surface with appropriate flexibility and hydrophobicity, cell growth and formation of three-dimensional tissues such as spheroids and three-dimensional cell aggregates are likely to proceed. Furthermore, when cell and culture medium are brought into contact with the surface of the base material containing the fluorine-containing polymer, and the other surface of the base material is contacted with an oxygen-containing gas such as air in addition to the top surface of the culture medium. Cell growth and formation of three-dimensional tissues such as spheroids and three-dimensional cell aggregates are likely to proceed.

  The present invention also relates to a method for culturing cells, comprising a step of culturing cells on the surface of the cell culture substrate, which is constituted by the resin composition.

The present invention further uses a substrate in which at least a part of one surface is made of a resin composition containing a fluorine-containing polymer, and the cell and the medium are in contact with the one surface of the substrate, Culturing the cells in a state where the other surface is in contact with the oxygen-containing gas,
The present invention relates to a method for culturing cells, wherein the fluorine-containing polymer is a polymer having a fluorine-containing aromatic ring and having an ether bond in the main chain.

  In the cell culture method of the present invention, three-dimensional culture of cells is possible by forming a surface serving as a cell scaffold with a predetermined resin composition.

  In a preferred embodiment of the above method, the culturing step is a step of three-dimensionally culturing cells.

  In a preferred embodiment of the above method, the step of three-dimensionally culturing the cell is a step of culturing the cell to form a spheroid or a three-dimensional cell aggregate.

  According to the method of the present invention, cell culture and formation of a three-dimensional tissue such as a spheroid are possible by a simple operation.

  In the present invention, “resin composition” can also be referred to as “resin”. Since the fluorine-containing polymer usually comprises a large number of fluorine-containing polymer molecules having different degrees of polymerization, a resin containing the fluorine-containing polymer is referred to as a “resin composition”. In the present invention, the “resin composition” or “resin” may or may not contain other components in an amount that does not impair the effects of the present invention. As another component, it can be the component used for a polymerization reaction of the quantity of the range which does not impair the effect of this invention, and a normal additive.

  In the cell culture substrate of the present invention, three-dimensional culture of cells becomes possible by constituting a surface that serves as a scaffold for cells with a predetermined resin composition.

  Further, according to the present invention, it is possible to form a three-dimensional tissue such as a spheroid or a three-dimensional cell aggregate from cells.

FIG. 1 is a schematic cross-sectional view of a film 1 made of a resin composition taken along a plane perpendicular to the main surface of the film 1 in Embodiment 1 of the cell culture substrate of the present invention. FIG. FIG. 2 is a cross-sectional view of a film 1 and a support 2 made of a resin composition taken along a plane perpendicular to the main surface of the film 1 in Embodiment 2 of the cell culture substrate of the present invention. FIG. FIG. 3 is a schematic diagram for explaining the positional relationship among the base material 10, the culture medium 2, the cells 3, and the outside air (oxygen-containing gas) 4 in the cell culture method of the present invention. One embodiment of the container for cell culture of the present invention is shown. 3 shows another embodiment of the cell culture vessel of the present invention. (A) It is a schematic diagram of the longitudinal cross-section of the container 100 for cell cultures. (B) It is a figure for demonstrating the method of performing cell culture using the container 100 for cell cultures. 4 shows another embodiment of the cell culture container of the present invention. (A) It is a perspective view of the container 100 for cell cultures. (B) It is the schematic diagram of the AA cross section of the container 100 for cell cultures. (C) It is a schematic diagram of the AA cross section of the container 100 for cell cultures of another embodiment. Fig. 4 shows a further embodiment of the cell culture vessel of the present invention. It is a photograph which shows the culture result of a rat primary hepatocyte. (A) shows the results of culture in a collagen type I-coated microplate 24 well (Asahi Glass Co., Ltd.) with a lid. (B) shows the culture results with the FPEK membrane of the present invention. It is a figure which shows the fixed_quantity | quantitative_assay result of the albumin in the culture solution of the culture | cultivation day 5 of a rat primary hepatocyte.

〔式中、Ｒ^３１、Ｒ^３２、Ｒ^３３及びＲ^３４のうち少なくとも１個がフッ素原子であり、Ｒ^３１、Ｒ^３２、Ｒ^３３及びＲ^３４がフッ素原子ではない場合、それぞれ独立して、水素原子（Ｈ）、シアノ基（ＣＮ）、置換基を有してもよい炭素原子数１〜１２のアルキル基、置換基を有してもよい炭素原子数１〜１２のアルコキシ基、置換基を有してもよい炭素原子数１〜１２のアルキルアミノ基、置換基を有してもよい炭素原子数１〜１２のアルキルチオ基、置換基を有してもよい炭素原子数６〜２０のアリール基、置換基を有してもよい炭素原子数６〜２０のアリールオキシ基、置換基を有してもよい炭素原子数６〜２０のアリールアミノ基、又は置換基を有してもよい炭素原子数６〜２０のアリールチオ基である。〕
で表される構造を繰り返し単位中に含み、主鎖にエーテル結合を有するポリマーである。 1. Fluorine-containing polymer The fluorine-containing polymer used in the present invention is a polymer having a fluorine-containing aromatic ring and having an ether bond in the main chain. Preferably, the following formula (I-1):

^Wherein at least one fluorine atom in R ^31, R ^{32, R 33} and ^{R 34,} when R ^31, R ^{32, R 33} and ^{R 34} is not a fluorine atom, each independently, hydrogen An atom (H), a cyano group (CN), an alkyl group having 1 to 12 carbon atoms which may have a substituent, an alkoxy group having 1 to 12 carbon atoms which may have a substituent, and a substituent; An optionally substituted alkylamino group having 1 to 12 carbon atoms, an optionally substituted alkylthio group having 1 to 12 carbon atoms, and an optionally substituted aryl having 6 to 20 carbon atoms Group, an aryloxy group having 6 to 20 carbon atoms which may have a substituent, an arylamino group having 6 to 20 carbon atoms which may have a substituent, or a carbon which may have a substituent An arylthio group having 6 to 20 atoms. ]
In a repeating unit, the polymer has an ether bond in the main chain.

In formula (I-1), it is preferable that at least two of R ³¹ , R ³² , R ³³ and R ³⁴ are fluorine atoms. Alternatively, all of R ³¹ , R ³² , R ³³ and R ³⁴ may be fluorine atoms.

  The fluorine content in the resin composition used in the present invention is 1 to 60% by mass, preferably 5 to 60% by mass, more preferably 10 to 60% by mass, and further preferably 15 to 50% by mass. Cells tend to form a three-dimensional tissue on the surface of the substrate composed of such a fluorine-containing resin composition.

〔式中、ｎは重合度を表し、ｍは０又は１の整数であり、Ｒ^４１は下記式（ＩＩ−３）：

（式中、ｐは０又は１の整数であり、Ｒ^４２は以下の構造：

のいずれかである。）で表される基である。〕
で示される含フッ素アリールエーテルケトン重合体である。 1.1. Specific example of fluorine-containing polymer (1)
Specific examples of the fluorine-containing polymer used in the present invention include the following formula (II-2):

[In the formula, n represents the degree of polymerization, m is an integer of 0 or 1, and R ⁴¹ represents the following formula (II-3):

(Wherein p is an integer of 0 or 1 and R ⁴² has the following structure:

One of them. ). ]
It is a fluorine-containing aryl ether ketone polymer shown by these.

  In the formula (II-2), the degree of polymerization n is specifically 2 to 5000, preferably 5 to 500. Furthermore, in the present invention, the fluorine-containing aryl ether ketone polymer may be composed of the same repeating unit or may be composed of different repeating units. In the latter case, the repeating unit is a block. Any of a shape or a random shape may be sufficient.

〔式中、ｎは重合度を表し、ｍは０又は１の整数であり、Ｒ^４１は上で定義した通りである。〕
で示される含フッ素アリールエーテルケトン重合体であると考えられる。 In addition, in this invention, although the manufacturing method of the fluorine-containing aryl ether ketone polymer shown by said Formula (II-2) is explained in full detail, from this description, the fluorine-containing aryl ether shown by Formula (II-2) The end of the ketone polymer is fluorine on the side of the benzene ring containing the fluorine atom and is hydrogen on the R ⁴¹ side, that is, the fluorine-containing aryl ether ketone polymer represented by the formula (II-2) is represented by the following formula (II -11):

[Wherein n represents the degree of polymerization, m is an integer of 0 or 1, and R ⁴¹ is as defined above. ]
It is thought that it is a fluorine-containing aryl ether ketone polymer shown by these.

〔式中、ｎは重合度を表す。〕
で示される含フッ素アリールエーテルケトン重合体となる。 In the above formula (II-2), when m is 0, the following formula (II-4):

[Wherein n represents the degree of polymerization. ]
It becomes the fluorine-containing aryl ether ketone polymer shown by these.

〔式中、ｎは重合度を表す。〕
で示される含フッ素アリールエーテルケトン重合体となる。 In the above formula (II-2), when m is 1 and p is 0, the following formula (II-5):

[Wherein n represents the degree of polymerization. ]
It becomes the fluorine-containing aryl ether ketone polymer shown by these.

〔式中、ｎは重合度を表し、Ｒ^４２は前記のとおりである。〕
で示される含フッ素アリールエーテルケトン重合体となる。なお、上記式（ＩＩ−６）では、ｎは、好ましくは２〜２０００、より好ましくは５〜２００である。 Furthermore, in the above formula (II-2), when m is 1 and p is 1, the following formula (II-6):

[Wherein n represents the degree of polymerization, and R ⁴² is as defined above. ]
It becomes the fluorine-containing aryl ether ketone polymer shown by these. In the above formula (II-6), n is preferably 2 to 2000, more preferably 5 to 200.

〔式中、Ｒ^４２は前記のとおりである。〕
で表される繰り返し単位を含むポリマーである。 That is, in a preferred embodiment, the fluorine-containing aryl ether ketone polymer represented by the formula (II-2) is represented by the following formula (II-1):

[Wherein, R ⁴² is as defined above. ]
It is a polymer containing the repeating unit represented by these.

で表される繰り返し単位を含むポリマーである。 In a particularly preferred embodiment, the fluorine-containing aryl ether ketone polymer represented by the formula (II-2) is represented by the following formula (II-12):

It is a polymer containing the repeating unit represented by these.

〔式中、ｑは０又は１の整数である。〕
で示される２，３，４，５，６−ペンタフルオロベンゾイル化合物を加熱することにより得られる。 Among these, the fluorine-containing aryl ether ketone polymers represented by the formulas (II-3) and (II-4) are represented by the following formula (II-9) in an organic solvent in the presence of a basic compound:

[Wherein q is an integer of 0 or 1; ]
It is obtained by heating a 2,3,4,5,6-pentafluorobenzoyl compound represented by

〔式中、Ｒ^４２は前記のとおりであり、ｎは重合度である。〕
で示される含フッ素アリールエーテルケトン重合体は、塩基性化合物の存在下で、有機溶媒中で、下記式（ＩＩ−８）：

で示される４，４’−ビス（２，３，４，５，６−ペンタフルオロベンゾイル）ジフェニルエーテル及び下記式（ＩＩ−１０）：

〔式中、Ｒ^４２は以下の構造：

のいずれかである。〕
で示される２価のフェノール化合物を加熱することよって、得られる。 In the above reaction, the reaction temperature is 30 to 250 ° C, preferably 50 to 200 ° C.
Moreover, following formula (II-6):

[Wherein, R ⁴² is as defined above, and n is the degree of polymerization. ]
A fluorine-containing aryl ether ketone polymer represented by the following formula (II-8) in an organic solvent in the presence of a basic compound:

4,4′-bis (2,3,4,5,6-pentafluorobenzoyl) diphenyl ether represented by formula (II-10):

[Wherein R ⁴² has the following structure:

One of them. ]
It can be obtained by heating a divalent phenol compound represented by the formula:

  In the above reaction, the reaction temperature is 20 to 150 ° C, preferably 50 to 120 ° C. At this time, by reacting at such a low temperature, side reactions can be suppressed and gelation of the polymer can be prevented. Moreover, although superposition | polymerization time changes with other reaction conditions, the raw material to be used, etc., Preferably it is 1-48 hours, More preferably, it is 2-24 hours. Furthermore, the polymerization reaction may be carried out under normal pressure or reduced pressure, but it is desirable to carry out under normal pressure from the viewpoint of equipment.

  Examples of the organic solvent used in the polymerization reaction include polar solvents such as N-methylpyrrolidinone, N-methyl-2-pyrrolidinone, N, N-dimethylacetamide and methanol, and toluene. These organic solvents may be used alone or in the form of a mixture of two or more.

  The concentration of the pentafluorobenzoyl diphenyl ether compound in the organic solvent is 5 to 50% by weight, preferably 10 to 30% by weight.

  When toluene or other similar solvent is used in the initial stage of the reaction, water produced as a by-product during phenoxide formation can be removed as an azeotrope of toluene regardless of the polymerization solvent.

  The basic compound used in the present invention acts to accelerate the polycondensation reaction by collecting hydrogen fluoride produced by the polycondensation reaction, and in the case of a polycondensation reaction with a divalent phenol compound. , Has the effect of changing the phenol compound to a more reactive anion.

  Examples of such basic compounds include potassium carbonate, lithium carbonate, and potassium hydroxide.

  In the present invention, the amount of the basic compound used is 0.5 in the case of the polymers of the formulas (II-3) and (II-4) with respect to 1 mol of the pentafluorobenzoyldiphenyl ether compound used. -10 mol, preferably 0.5-5 mol, or in the case of the polymer of formula (II-5), preferably 1-20 mol, preferably 1-20 mol, per mol of pentafluorobenzoyl diphenyl ether compound used. Is 1 to 10 moles.

  Examples of the divalent phenol compound used in the present invention include 2,2-bis (4-bidoxyphenyl) -1,1,1,3,3,3-hexafluoropropane (hereinafter referred to as “6FBA”). ), Bisphenol A (hereinafter referred to as “BA”), 9,9-bis (4-hydroxyphenyl) fluorene (hereinafter referred to as “HF”), bisphenol F (hereinafter referred to as “BF”), hydroquinone (hereinafter referred to as “BF”). , "HQ") and resorcinol (hereinafter referred to as "RS"). The amount of the divalent phenol compound used is 0.8 to 1.2 mol, preferably 0 with respect to 1 mol of 4,4′-bis (2,3,4,5,6-pentafluorobenzoyl) diphenyl ether. .9 to 1.1 moles.

  For example, 4,4′-bis (2,3,4,5,6-pentafluorobenzoyl) diphenyl ether is reacted with 6FBA as a divalent phenol compound in an organic solvent in the presence of a basic compound. By this, the fluorine-containing aryl ether ketone polymer containing the repeating unit represented by the above formula (II-12) can be produced.

  After completion of the polymerization reaction, the solvent is removed from the reaction solution by evaporation or the like, and the distillate is washed as necessary to obtain a desired polymer. Alternatively, the polymer may be obtained by precipitating the polymer as a solid by adding the reaction solution in a solvent having low polymer solubility, and separating the precipitate by filtration.

1.2. Specific example of fluorine-containing polymer (2)
Another specific example of the fluorine-containing polymer is represented by the following formula (III-1):

〔式中、ｍ’及びｎ’は、同一又は異なって、ベンゼン環に付加しているフッ素原子の数を表し、１〜４の整数である。Ｒ^５１及びＲ^５２は、同一又は異なって、炭素数１〜１５０の２価の有機基を表す。ｐは、重合度を表す。〕
で表される繰り返し単位を必須とする含フッ素アリールエステル重合体である。

[Wherein, m ′ and n ′ are the same or different and represent the number of fluorine atoms added to the benzene ring, and are integers of 1 to 4. R ⁵¹ and R ⁵² are the same or different and represent a divalent organic group having 1 to 150 carbon atoms. p represents the degree of polymerization. ]
It is a fluorine-containing aryl ester polymer which has the repeating unit represented by these.

  The fluorine-containing aryl ester polymer used in the present invention may contain other repeating units as long as the repeating unit represented by the above formula (III-1) is essential, but the above formula ( It is preferable that the repeating unit represented by III-1) is a main component of the repeating unit constituting the fluorine-containing aryl ester polymer. In the fluorine-containing aryl ester polymer, the structure of the repeating unit represented by the above formula (III-1) may be the same or different. Any form such as a block form or a random form may be used.

In the repeating unit represented by the above formula (III-1), the (—O—R ⁵² —O—) moiety is an ortho position, a meta position, a para position with respect to the carbon bonded to the ester of the benzene ring. These may be bonded to any carbon, but those bonded to the ortho or para position are preferred. In the fluorine-containing aryl ester polymer used in the present invention, the benzene ring having a fluorine atom has a form in which part or all of four hydrogen atoms are substituted with fluorine atoms, and the hydrogen of the benzene ring The atom may be substituted by other substituents such as a halogen atom other than a fluorine atom or a substituent having an alkyl chain. Therefore, the total of hydrogen atoms, fluorine atoms, halogen atoms other than fluorine atoms, and other substituents in one benzene ring is 4.

R ⁵¹ and R ⁵² are the same or different and represent a divalent organic group having 1 to 150 carbon atoms, and the divalent organic group is preferably an organic group having 1 to 50 carbon atoms. More preferably, it is any one of groups represented by the following formulas (8-1) to (8-19).

In the above formulas (8-1) to (8-19), Y ¹ , Y ¹ ′, Y ² , Y ² ′, Y ³ and Y ⁴ are the same or different and each represents a substituent and represents one benzene ring Has 0 to 4 Y ¹ , Y ² , Y ³ and Y ⁴ , 0 to 3 Y ¹ ′ and Y ² ′ as substituents. Examples of the substituent represented by Y ¹ , Y ¹ ′, Y ² , Y ² ′, Y ^3, and Y ⁴ include a halogen atom, an optionally substituted alkyl group, an alkoxy group, and an alkylamino group. Group, alkylthio group, aryl group, aryloxy group, arylamino group, arylthio group and the like.

  In the fluorine-containing aryl ester polymer used in the present invention, the benzene ring of the group represented by the above formulas (8-1) to (8-19) has one or more substituents, and the substituents are carbon. It is a number 1-30, It is preferable that it is an alkyl group, an alkoxy group, etc. which may have a substituent, or it is preferable that a benzene ring does not have a substituent. More preferably, the benzene ring has no substituent. That is, a group represented by the following formulas (9-1) to (9-19) is preferable.

〔式中、ｍ’及びｎ’は、同一又は異なって、ベンゼン環に付加しているフッ素原子の数を表し、１〜４の整数である。Ｒ^５２は、同一又は異なって、炭素数１〜１５０の２価の有機基を表す。ｐは、重合度を表す。〕
で表される繰り返し単位及び／又は下記式（ＩＩＩ−３）；

〔式中、ｍ’及びｎ’は、同一又は異なって、ベンゼン環に付加しているフッ素原子の数を表し、１〜４の整数である。Ｒ^５２は、同一又は異なって、炭素数１〜１５０の２価の有機基を表す。ｐは、重合度を表す。〕
で表される繰り返し単位を必須とするものであることが好ましい。 In the fluorine-containing aryl ester polymer used in the present invention, in the formula (III-1), the structure of R ⁵¹ is any one of the above (9-6) or (9-18), and a benzene ring. Are preferably those having no substituent. That is, the fluorine-containing aryl ester polymer of the present invention has the following formula (III-2):

[Wherein, m ′ and n ′ are the same or different and represent the number of fluorine atoms added to the benzene ring, and are integers of 1 to 4. R ⁵² is the same or different and represents a divalent organic group having 1 to 150 carbon atoms. p represents the degree of polymerization. ]
And / or the following formula (III-3):

[Wherein, m ′ and n ′ are the same or different and represent the number of fluorine atoms added to the benzene ring, and are integers of 1 to 4. R ⁵² is the same or different and represents a divalent organic group having 1 to 150 carbon atoms. p represents the degree of polymerization. ]
It is preferable that the repeating unit represented by is essential.

  The number average molecular weight (Mn) of the fluorine-containing aryl ester polymer may be appropriately set according to the required characteristics, but is preferably 1000 or more and 1000000 or less. More preferably, it is 3000 or more and 500000 or less. The number average molecular weight can be measured using GPC (manufactured by Tosoh Corporation, HLC-8120GPC) using polystyrene as a standard sample and THF as a developing solvent.

  Although the manufacturing method in particular of the fluorine-containing aryl ester polymer represented by the said Formula (III-1) is not restrict | limited, The manufacturing method including the process of superposing | polymerizing a fluorine-containing ester compound and a dihydroxy compound is preferable. From the viewpoint of reaction efficiency, this step is preferably performed in the presence of a basic catalyst.

〔式中、ｍ及びｎは、同一又は異なって、ベンゼン環に付加しているフッ素原子の数を表し、１〜５の整数である。Ｒ^５１は、炭素数１〜１５０の２価の有機基を表す。〕
で表される含フッ素エステル化合物と下記式（ＩＩＩ−５）：

〔式中、Ｒ^５２は、炭素数１〜１５０の２価の有機基を表す。〕
で表されるジヒドロキシ化合物とを塩基性触媒存在下で重合する工程を含む製造方法により製造されることが好ましい。 That is, the fluorine-containing aryl ester polymer represented by the above formula (III-1) is represented by the following formula (III-4):

[Wherein, m and n are the same or different and represent the number of fluorine atoms added to the benzene ring, and are integers of 1 to 5. R ⁵¹ represents a divalent organic group having 1 to 150 carbon atoms. ]
And a fluorine-containing ester compound represented by the following formula (III-5):

[Wherein, R ⁵² represents a divalent organic group having 1 to 150 carbon atoms. ]
It is preferable to manufacture by the manufacturing method including the process of superposing | polymerizing in the presence of a basic catalyst with the dihydroxy compound represented by these.

  Since the fluorine-containing ester compound represented by the above formula (III-4) has high reactivity, when a polymer is produced from this fluorine-containing ester compound as a raw material as in the above production method, a homogeneous system is used. Various polymerization methods can be used regardless of the polymerization method, such as polymerization in a polymer, interfacial polymerization, etc., and it is also polymerized under conditions of 150 ° C. or lower, which is a lower temperature than conventional polymerization reactions using fluorine-containing compounds. It is possible.

In the above production method, the structure of the structure derived from the dihydroxy compound (—O—R ⁵² —O—) is in any of the ortho, meta, and para positions with respect to the carbon to which the ester group of the benzene ring is bonded. Although it may be bonded to carbon, those bonded to the ortho position or para position are preferred. In addition, if two or more parts derived from a dihydroxy compound are bonded to one benzene ring, a crosslinked structure may be formed. If the crosslinked structure has a crosslinked structure, the resulting polymer will gel, Those with less are preferred. In the above production method, the ease of formation of a cross-linked structure differs depending on the reaction temperature and reaction time, the type and concentration of the solvent and basic catalyst used, the raw material charging order, the amount of water in the reaction system, etc. It becomes possible to suppress the formation of a crosslinked structure.

  In the polycondensation reaction in the above production method, it is preferable to appropriately set the ratio of the dihydroxy compound used as the raw material and the fluorinated ester compound from the viewpoint of effective utilization of the reaction raw material and improvement in the yield of the product, It is preferable to use 0.8 to 1.2 mol of the dihydroxy compound with respect to 1 mol of the fluorinated ester compound. More preferably, 0.9 to 1.1 mol of dihydroxy compound is used.

  The reaction temperature of the polycondensation reaction in the above production method is preferably 0 to 100 ° C, more preferably 10 to 80 ° C. The reaction time is preferably 1 to 40 hours, more preferably 1 to 30 hours. Furthermore, the above reaction may be performed under reduced pressure, normal pressure, or increased pressure, but it is preferable to perform the reaction under normal pressure in consideration of equipment and the like.

  In the polycondensation reaction in the above production method, various solvents can be used because the fluorine-containing ester compound is excellent in solubility in a solvent, and nitriles such as acetonitrile and benzonitrile; nitrobenzene and nitromethane Nitros such as acetone, methyl isobutyl ketone (MIBK), methyl ethyl ketone (MEK), cyclohexanone, etc .; halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, chloroethane, dichloroethane, trichloroethane, tetrachloroethane; Aromatic hydrocarbons such as benzene, toluene, xylene; hydrocarbons such as pentane, hexane, cyclohexane, heptane; diethyl ether, isopropyl ether, tetrahydrofuran (THF), dioxane, diphenyl ether Ethers such as tellurium, benzyl ether, tert-butyl ether; esters such as methyl formate, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate; N-methyl-2-pyrrolidinone (NMP), dimethylformamide (DMF) ), Dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc), and the like can be used. These may be used alone or in combination of two or more. Among these, acetone, acetonitrile, methyl isobutyl ketone (MIBK), methyl ethyl ketone (MEK), N-methyl-2-pyrrolidinone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and dimethylacetamide (DMAc) are preferable. . The amount of the solvent may be an amount that allows the above reaction to proceed efficiently, but is preferably an amount such that the fluorine-containing ester compound is 1 to 50% by mass in the solvent, more preferably 1 to 30%. The amount is such that the mass% is present.

  As the basic compound used in the polycondensation reaction in the above production method, it acts to promote the polycondensation reaction by collecting hydrogen fluoride produced by the polycondensation reaction. What has the effect | action which changes to the highly reactive anion is suitable, for example, 1 type, such as calcium carbonate, calcium hydroxide, potassium fluoride, tributylamine, pyridine, potassium carbonate, lithium carbonate, potassium hydroxide, triethylamine, or Two or more are preferred. As the usage-amount of such a basic compound, 0.5-20 mol is preferable with respect to 1 mol of fluorine-containing ester compounds to be used. More preferably, it is 0.8-10 mol.

  After completion of the polycondensation reaction, the solvent in the reaction solution is removed by evaporation or the like, and the distillate is washed as necessary, whereby the fluorine-containing aryl having the repeating unit represented by the above formula (III-1) An ester polymer will be obtained. It can also be obtained by adding the reaction solution to a solvent having low polymer solubility to precipitate the fluorinated aryl ester polymer as a solid and separating the precipitate by filtration.

  Since the fluorine-containing aryl ester polymer of the present invention is excellent in solubility in a solvent, it can be molded into various shapes such as films and fibers. The molded product containing the fluorine-containing aryl ester polymer of the present invention has high moldability due to excellent solvent solubility, heat resistance, low moisture absorption, transparency, weather resistance, water repellency and It has excellent electrical characteristics.

〔式中、Ｒ^３１は、置換基を有してもよい炭素原子数１〜１２のアルキル基、置換基を有してもよい炭素原子数１〜１２のアルコキシ基、置換基を有してもよい炭素原子数１〜１２のアルキルアミノ基、置換基を有してもよい炭素原子数１〜１２のアルキルチオ基、置換基を有してもよい炭素原子数６〜２０のアリール基、置換基を有してもよい炭素原子数６〜２０のアリールオキシ基、置換基を有してもよい炭素原子数６〜２０のアリールアミノ基又は置換基を有してもよい炭素原子数６〜２０のアリールチオ基を表し；Ｒ^６１は、２価の有機基を表し；並びにｎは重合度を表す。〕
で示されるポリシアノアリールエーテルである。 1.3. Specific example of fluorine-containing polymer (3)
Another specific example of the fluorine-containing polymer that can be used in the present invention is represented by the following formula (IV-1):

[Wherein R ³¹ has an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted alkoxy group having 1 to 12 carbon atoms, and a substituent. An alkylamino group having 1 to 12 carbon atoms, an alkylthio group having 1 to 12 carbon atoms which may have a substituent, an aryl group having 6 to 20 carbon atoms which may have a substituent, and a substituent An aryloxy group having 6 to 20 carbon atoms which may have a group, an arylamino group having 6 to 20 carbon atoms which may have a substituent or a carbon atom having 6 to 20 carbon atoms which may have a substituent 20 represents an arylthio group; R ⁶¹ represents a divalent organic group; and n represents the degree of polymerization. ]
It is polycyanoaryl ether shown by these.

In the formula (IV-1), R ³¹ is an optionally substituted alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert -Butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and 2-ethylhexyl, preferably methyl, ethyl, propyl and butyl; optionally having 1 to 1 carbon atoms 12 alkoxy groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentyloxy, hexyloxy, 2-ethylhexyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, furfuryloxy and allyloxy, preferably Is Methoxy, ethoxy, propoxy, isopropoxy and butoxy; optionally substituted alkylamino groups having 1 to 12 carbon atoms such as methylamino, ethylamino, dimethylamino, diethylamino, propylamino, n-butylamino , Sec-butylamino and tert-butylamino, preferably methylamino, ethylamino, dimethylamino and diethylamino; optionally substituted alkylthio groups having 1 to 12 carbon atoms such as methylthio, ethylthio, propylthio and n-butylthio, sec-butylthio, tert-butylthio and iso-propylthio, preferably methylthio, ethylthio and propylthio; aryl groups having 6 to 20 carbon atoms which may have a substituent, for example, phenyl, benzyl, phenethyl , O-, m- or p-tolyl, 2,3- or 2,4-xylyl, mesityl, naphthyl, anthryl, phenanthryl, biphenylyl, benzhydryl, trityl and pyrenyl, preferably phenyl and o-, m- and p- Tolyl; optionally substituted aryloxy group having 6 to 20 carbon atoms such as phenoxy, benzyloxy, hydroxybenzoic acid and esters thereof (for example, methyl ester, ethyl ester, methoxyethyl ester, ethoxyethyl) Groups derived from esters, furfuryl esters and phenyl esters; hereinafter the same), naphthoxy, o-, m- or p-methylphenoxy, o-, m- or p-phenylphenoxy, phenylethynylphenoxy, and cresotinic acid and Derived from its esters Groups, preferably phenoxy and naphthoxy; optionally substituted arylamino groups having 6 to 20 carbon atoms, such as anilino, o-, m- or p-toluidino, 1,2- or 1,3- Xylidino, o-, m- or p-methoxyanilino and groups derived from anthranilic acid and its esters, preferably anilino and o-, m- or p-toluidino; or the number of carbon atoms which may have a substituent It represents 6 to 20 arylthio groups such as phenylthio, phenylmethanethio, o-, m- or p-tolylthio and groups derived from thiosalicylic acid and its esters, preferably phenylthio. Among these, an aryloxy group, an arylthio group and an arylamino group which may have a substituent are preferable, and phenoxy, phenylthio and anilino are most preferable as R ³¹ .

Further, in the above formula (IV-1), R ³¹ can be used when it represents a substituted alkyl group, alkoxy group, alkylamino group, alkylthio group, aryl group, aryloxy group, arylamino group or arylthio group. The substituent can be appropriately selected according to the desired properties of the target product and is not particularly limited. For example, the alkyl group has 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl. , Isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl; halogen atoms such as fluorine, chlorine, bromine and iodine; cyano group, nitro group and A carboxyester group etc. are mentioned. Of these, methyl and carboxyester groups are preferred.

Further, in the above formula (IV-1), R ⁶¹ represents a divalent organic group, and examples thereof include a group represented by the following formula.

で示される２価の有機基がＲ^６１として好ましく、特に下記式：

で示される２価の有機基がＲ^６１として好ましい。 Of these, the following formula:

A divalent organic group represented by is preferable as R ⁶¹ , and in particular, the following formula:

Is preferably R ⁶¹ .

  Furthermore, in said formula (IV-1), n represents a polymerization degree, and is specifically 5-1000, Preferably it is 10-500. The polycyanoaryl ether used in the present invention may be composed of the same repeating unit of the structural unit of the above formula (IV-1) or may be composed of different repeating units. In some cases, the repeating unit may be either block or random.

で示されるポリマーであると考えられる。 Moreover, although the manufacturing method of the polycyanoaryl ether of this invention is explained in full detail below, from this description, the terminal of the polycyanoaryl ether represented by the formula (IV-1) is a fluorine atom containing a fluorine atom. When the oxygen atom (R ⁶¹ ) side is a hydrogen atom, that is, the polycyanoaryl ether represented by the formula (IV-1) is represented by the following formula (IV-4):

It is thought that it is a polymer shown by.

で示されるテトラフルオロベンゾニトリル誘導体を、下記式（ＩＶ−３）：

で示されるジヒドロキシ化合物と塩基性触媒の存在下で重合することによって、製造される。この際、上記式（ＩＶ−２）におけるＲ^３１及び上記式（ＩＶ−３）におけるＲ^６１の定義は、上記式（ＩＶ−１）におけるＲ^３１及びＲ^６１の定義と同様である。 The polycyanoaryl ether of the present invention has the following formula (IV-2):

A tetrafluorobenzonitrile derivative represented by the following formula (IV-3):

It is manufactured by polymerizing in the presence of a basic catalyst and a dihydroxy compound represented by At this time, the definition of R ³¹ in the above formula (IV-2) and R ^{61 in} the above formula (IV-3) is the same as the definition of R ³¹ and R ⁶¹ in the above formula (IV-1).

In the present invention, the tetrafluorobenzonitrile derivative of the formula (IV-2) can be produced by a known method. For example, the formula: R ³¹ H [wherein R ³¹ is as defined in the above formula (IV-1) The same as above] is reacted with 2,3,4,5,6-pentafluorobenzonitrile (also referred to herein as “PFBN”) in the presence of a basic compound in an organic solvent. Obtained by.

In the above reaction, the compound represented by the formula: R ³¹ H and PFBN may be used as a single compound or a mixture of two or more compounds represented by the formula: R ³¹ H and / or PFBN, respectively. However, in consideration of the purification process and the physical properties of the polymer, it is preferably used as a single compound. In the latter case, the total number of moles of plural or single PFBN used may be equal to or substantially equal to the total number of moles of the compound represented by the plural or single formula: R ³¹ H. Specifically, the amount of the compound represented by the formula: R ³¹ H is preferably 0.1 to 5 mol, more preferably 0.5 to 2 mol, relative to 1 mol of PFBN.

  Examples of the organic solvent that can be used in the above reaction include polar solvents such as N-methyl-2-pyrrolidinone, N, N-dimethylacetamide, acetonitrile, benzonitrile, nitrobenzene, nitromethane, and methanol; and these polar solvents and toluene, Examples thereof include a mixed solvent with a nonpolar solvent such as xylene. These organic solvents may be used alone or in the form of a mixture of two or more. Moreover, the density | concentration of PFBN in an organic solvent is 1-40 mass%, Preferably, it is 5-30 mass%. In this case, when toluene or other similar solvent is used in the initial stage of the reaction, water produced as a by-product during the reaction can be removed as an azeotrope of toluene regardless of the polymerization solvent.

  Moreover, it is desirable that the basic compound used in the above reaction acts to collect hydrogen fluoride generated to promote the reaction. Examples of such basic compounds include potassium carbonate, calcium carbonate, potassium hydroxide, calcium hydroxide, potassium fluoride, triethylamine, tributylamine, and pyridine. Under the present circumstances, the usage-amount of a basic compound is 0.1-5 mol with respect to 1 mol of PFBN used, Preferably it is 0.5-2 mol.

Furthermore, the reaction conditions in the above reaction are not particularly limited as long as the reaction between the compound represented by R ¹ H and PFBN proceeds efficiently. For example, the reaction is preferably performed by stirring the reaction system. While maintaining the state, the reaction is usually performed at a temperature of 20 to 180 ° C, preferably 40 to 160 ° C. Moreover, although reaction time changes with other reaction conditions, the raw material to be used, etc., it is 1-48 hours normally, Preferably it is 2-24 hours. Furthermore, the reaction may be carried out under normal pressure or under reduced pressure, but it is desirable to carry out under normal pressure from the viewpoint of equipment. The product obtained by such a reaction is obtained by pouring distilled water into the reaction mixture and extracting with an extractant such as dichloromethane, dichloroethane or carbon tetrachloride, and then separating the organic layer from the extract and distilling the extractant. It is obtained by leaving. Furthermore, if necessary, this product may be obtained as crystals by recrystallization from methanol or ethanol.

  The tetrafluorobenzonitrile derivative of the formula (IV-2) synthesized in this way is subjected to polymerization in the presence of a dihydroxy compound of the formula (IV-3) and a basic catalyst as described above. To produce the desired polycyanoaryl ether of formula (IV-1). At this time, the tetrafluorobenzonitrile derivative of the formula (IV-2) may be used after being subjected to a purification process such as extraction, recrystallization, chromatography and distillation as described above or without performing the purification process. Although it may be used, it is preferably used after purification in consideration of the yield of the next step.

  The dihydroxy compound of formula (IV-3) used in the above reaction is selected according to the structure of the polycyanoaryl ether of formula (IV-1) which is the target product. The dihydroxy compound of the formula (IV-3) preferably used includes 2,2-bis (4-bidoxyphenyl) -1,1,1,3,3,3-hexa, as shown below. Fluoropropane (hereinafter referred to as “6FBA”), 4,4′-dihydroxydiphenyl ether (hereinafter referred to as “DPE”), hydroquinone (hereinafter referred to as “HQ”), bisphenol A (hereinafter referred to as “BA”), 9, 9-bis (4-hydroxyphenyl) fluorene (hereinafter referred to as “HF”), phenolphthalein (hereinafter referred to as “PP”), 9,9-bis (3-methyl-4-hydroxyphenyl) fluorene (hereinafter referred to as “PP”). "MHF"), 1,4-bis (hydroxyphenyl) cyclohexane (hereinafter referred to as "CHB"), and 4,4'-dihydroxybiphe Le (hereinafter referred to as "BP"), and the like.

  In the above reaction, the tetrafluorobenzonitrile derivative of the formula (IV-2) and the dihydroxy compound of the formula (IV-3) may each be used as a single compound or two or more of the formulas (IV-2) May be used in the form of a mixture of a tetrafluorobenzonitrile derivative and / or a dihydroxy compound of the formula (IV-3), but it should be used as a single compound in consideration of the purification process and the physical properties of the polymer. Is preferred. In the latter case, the total number of moles of the plural or single tetrafluorobenzonitrile derivative of the formula (IV-2) used is the plural or single dihydroxy compound of the formula (IV-3). It is preferable that the amount of the dihydroxy compound of the formula (IV-3) is used with respect to 1 mol of the tetrafluorobenzonitrile derivative of the formula (IV-2). 0.1 to 5 mol, preferably 1 to 2 mol.

  The above reaction may be performed in an organic solvent or in the absence of a solvent, but is preferably performed in an organic solvent. In the former case, examples of organic solvents that can be used include polar solvents such as N-methyl-2-pyrrolidinone, N, N-dimethylacetamide, acetonitrile, benzonitrile, nitrobenzene, nitromethane, and methanol; and these polar solvents and toluene. And a mixed solvent with a nonpolar solvent such as xylene. These organic solvents may be used alone or in the form of a mixture of two or more. The concentration of the tetrafluorobenzonitrile derivative of the formula (IV-2) in the organic solvent is 1 to 50% by mass, preferably 5 to 20% by mass. In this case, when toluene or other similar solvent is used in the initial stage of the reaction, water produced as a by-product during the reaction can be removed as an azeotrope of toluene regardless of the polymerization solvent.

  In the present invention, the reaction of the tetrafluorobenzonitrile derivative of formula (IV-2) and the dihydroxy compound of formula (IV-3) must be carried out in the presence of a basic catalyst. The basic catalyst preferably has a function of converting the dihydroxy compound of the formula (IV-3) into a more reactive anion so as to promote the polycondensation reaction by the dihydroxy compound of the formula (IV-3). Examples include potassium carbonate, calcium carbonate, potassium hydroxide, calcium hydroxide, and potassium fluoride. The amount of the basic catalyst used is not particularly limited as long as the reaction between the tetrafluorobenzonitrile derivative of formula (IV-2) and the dihydroxy compound of formula (IV-3) can proceed satisfactorily. Although there is not, it is 0.1-5 mol normally with respect to 1 mol of tetrafluorobenzonitrile derivatives of a formula (IV-2), Preferably it is 0.5-2 mol.

  Furthermore, the reaction conditions in the above polymerization reaction are not particularly limited as long as the reaction between the tetrafluorobenzonitrile derivative of formula (IV-2) and the dihydroxy compound of formula (IV-3) proceeds efficiently. However, for example, the polymerization temperature is preferably 200 ° C. or lower, more preferably 20 to 150 ° C., and most preferably 40 to 100 ° C. By reacting at such a low temperature, side reactions can be suppressed and gelation of the polymer can be prevented without requiring special equipment. Moreover, although superposition | polymerization time changes with other reaction conditions, the raw material to be used, etc., Preferably it is 1-48 hours, More preferably, it is 2-24 hours. Furthermore, the polymerization reaction may be carried out under normal pressure or reduced pressure, but it is desirable to carry out under normal pressure from the viewpoint of equipment.

  After completion of the polymerization reaction, the solvent is removed from the reaction solution by evaporation or the like, and the distillate is washed as necessary to obtain a desired polymer. Alternatively, the polymer may be obtained by adding the reaction solution in a solvent having low polymer solubility to precipitate the polymer as a solid and separating the precipitate by filtration.

1.4. Chemical and physical characteristics of polymer-containing resin composition
1.4.1. Oxygen gas permeability coefficient In order for the cell culture substrate of the present invention to have high oxygen gas permeability, it is preferable to use a resin composition containing a fluorine-containing polymer having a high oxygen gas permeability coefficient. Specifically, the oxygen gas permeability coefficient of the resin composition is preferably 0.10 × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or more, more preferably 0.50. × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or more, more preferably 1.0 × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or more, More preferably 1.5 × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or more, more preferably 2.0 × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or more, more preferably 2.5 × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or more, more preferably 3.0 × 10 ⁻¹⁰ cm ³ (STP) · cm / (cm ² · s · cmHg) or less Above. The higher the oxygen gas permeation coefficient of the resin composition containing the fluorine-containing polymer, the easier the oxygen supply to the cultured cells proceeds. The upper limit value of the oxygen gas permeability coefficient of the resin composition is not particularly limited, but is usually 2.0 × 10 ⁻⁸ cm ³ (STP) · cm / (cm ² · s · cmHg) or less, preferably 1.5. × 10 ⁻⁸ cm ³ (STP) · cm / (cm ² · s · cmHg) or less.

1.4.2. Water contact angle The water contact angle of the surface constituted by the resin composition containing the fluorine-containing polymer (processed into a film, film, plate, etc.) in the cell culture substrate of the present invention is preferably Is 70 ° or more, more preferably 73 ° or more, still more preferably 75 ° or more, preferably 115 ° or less, more preferably 112 ° or less, still more preferably 110 ° or less. When the water contact angle is within this range, the cells are likely to adhere to the substrate surface with an appropriate strength, and the cells can form a three-dimensional tissue using the surface as a scaffold. The contact angle can be calculated by measuring the contact angle with water at a temperature of 25 ° C. using an automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500).

1.4.3. Tensile modulus The resin composition containing the fluorine-containing polymer is also preferably excellent in flexibility. In one embodiment, the resin composition containing the fluorine-containing polymer is excellent in flexibility due to the polymerization unit of the fluorine-containing polymer having an ether bond. The flexibility can be evaluated by a tensile elastic modulus. For example, the tensile modulus can be 2 GPa or less. Thus, the form in which the resin composition has a tensile modulus of 2 GPa or less is also a preferred form of the present invention. Cells tend to form a three-dimensional structure on the surface constituted by a resin composition having flexibility in which the tensile modulus is in this range. The tensile elastic modulus of the resin composition is more preferably 1.8 GPa or less, and still more preferably 1.5 GPa or less. Although the minimum of a tensile elasticity modulus is not specifically limited, Preferably it is 0.3 GPa or more, More preferably, it is 0.5 GPa or more. The tensile modulus (GPa) can be measured by a dynamic viscoelasticity measuring method known in the art.

2. Cell Culture Substrate The cell culture substrate of the present invention is characterized in that at least a part of the surface is composed of a resin composition containing the fluorine-containing polymer.

  The form of the substrate for cell culture is not particularly limited as long as it is a member used for cell culture and having a surface serving as a scaffold for cell proliferation. For example, a substrate for cell culture in the form of a film or plate is used for cell culture by placing a medium containing cells on one surface, or for culturing such a substrate as a single or multiwell plate. It can be housed and fixed in various cell culture containers such as plates, petri dishes, dishes, flasks, culture bags, etc., and cell culture can be carried out by adding a medium containing cells to the container. In addition, the cell culture substrate itself may be in the form of various cell culture containers such as a culture plate such as a single or multi-well plate, a culture dish, a culture dish, a flask, or a culture bag. The culture bag can be used for culturing floating cells, stem cells, etc. in a floating state.

  The surface constituted by the resin composition is part or all of the surface of the cell culture substrate that comes into contact with the medium containing cells during cell culture, preferably the surface of the cell culture substrate Among these, a part or all of the surface located vertically below the medium containing cells during cell culture. The entire surface of the cell culture substrate may be composed of the resin composition. In the portion of the substrate for cell culture whose surface is constituted by the resin composition, it is sufficient that the outermost surface used by the cells as a scaffold during cultivation is constituted by the resin composition, and the thickness direction of the portion The material at a position away from the outermost surface along the line is not particularly limited. That is, the cell culture substrate of the present invention only needs to be provided with a layer composed of the resin composition on at least part or all of the surface that comes into contact with the medium containing cells during cell culture. For example, as in Embodiment 1 shown in FIG. 1, the portion of the cell culture substrate that includes the resin composition on the surface S is composed of the resin composition not only on the surface but also in the entire thickness direction. Alternatively, as in the second embodiment shown in FIG. 2, the film 1 of the resin composition is formed on and in the vicinity of the outermost surface S that serves as a scaffold for cells during culture, and the outermost surface of the film 1 On the side opposite to S, a support 2 made of any material may be disposed.

  A preferred embodiment of the substrate for cell culture of the present invention is a film-like substrate for cell culture (Embodiment 1) constituted by the resin composition, or a support, and a surface integrated with the support. It is the base material for cell cultures provided with the film comprised by the said resin composition which covers at least one part (Embodiment 2). That is, as shown in FIG. 1, the cell culture substrate 10 according to Embodiment 1 includes a film 1 composed of the resin composition. As shown in FIG. 2, the cell culture substrate 10 according to Embodiment 2 includes a film 1 and a support 2 that are formed of the resin composition. In either embodiment, the film constituted by the resin composition can be formed by the same method. In Embodiment 2, the support may be a film-like, porous support, mesh-like support or the like, and various cells such as plates, culture dishes, dishes, single or multiwell plates, flasks, etc. It can be made into arbitrary shapes which can be used for a cell culture use, such as the shape of a container for culture.

  In one embodiment, the cell culture substrate of the present invention is preferably a cell culture substrate comprising a film composed of a resin composition containing the fluorine-containing polymer on at least a part of the surface.

In the present invention, “oxygen gas permeability coefficient” and “oxygen gas permeability” refer to values measured by a method according to JIS K7126-1 (differential pressure method) appendix 2, respectively. “Oxygen gas permeability coefficient” and “oxygen gas permeability” are both values obtained by converting values measured at 25 ° C. and a relative humidity of approximately 0% into a standard state of 0 ° C. and 1 atm. Specifically, the following measurement conditions can be employed.
Test method: Differential pressure method (according to JIS K7126-1 Annex 2)
Detector: Gas chromatograph (Thermal conductivity detector: TCD)
Test differential pressure: 1 atm
Test gas: Oxygen gas (Dry state (relative humidity approximately 0%))
Test conditions: 25 ° C ± 2 ° C
Transmission area: 1.52 × 10 ⁻³ m ²
Equipment: Differential pressure type gas / vapor permeability measuring device (GTR-30XAD2, G2700T · F)
GTR Tech Co., Ltd./Yanaco Technical Science Co., Ltd.

  When the cell culture substrate is composed of a plurality of layers as in the example shown in FIG. 3, the total oxygen gas permeability of the cell culture substrate may be obtained by direct measurement, or the oxygen in each layer The oxygen gas permeability of the entire substrate may be calculated from the gas permeability.

The cell culture substrate used in the present invention has an oxygen gas permeability of 219 cm ³ (STP) / (m ² · 24 h · atm) or more. When the cell culture substrate of the present invention has a high oxygen gas permeability of 219 cm ³ (STP) / (m ² · 24 h · atm) or more, cells are placed on the surface of the substrate containing the resin composition. When culturing, oxygen is easily supplied, and cell growth, formation of a three-dimensional tissue by the cell, and tissue growth are likely to proceed. The oxygen gas permeability of the cell culture substrate of the present invention is more preferably at least 1094 cm ³ (STP) / (m ² · 24 h · atm), more preferably 2189 cm ³ (STP) / (m ² · 24 h · atm). ) Or more, more preferably 3283 cm ³ (STP) / (m ² · 24 h · atm) or more, more preferably 4378 cm ³ (STP) / (m ² · 24 h · atm) or more, more preferably 5472 cm ³ (STP) / (M ² · 24h · atm) or more, more preferably 6566 cm ³ (STP) / (m ² · 24h · atm) or more. The higher the oxygen gas permeability of the cell culture substrate of the present invention is, the easier it is to supply oxygen to the cultured cells. The upper limit value of the oxygen gas permeability of the cell culture substrate of the present invention is not particularly limited, but is usually 437760 cm ³ (STP) / (m ² · 24 h · atm) or less, preferably 328320 cm ³ (STP) / (m ² · 24h · atm) or less.

  The thickness (not including the thickness of the support) of the layer (also referred to as “film” or “membrane”) composed of the resin composition containing a fluorine-containing polymer in the cell culture substrate of the present invention is Although it can be adjusted as appropriate so that the overall material has an appropriate oxygen gas permeability, it is typically preferably 0.1 μm or more and 5 mm or less, more preferably 0.5 μm or more and 3 mm or less. It is preferably 1 μm or more and 2 mm or less, more preferably 5 μm or more and 1 mm or less.

  When the cell culture substrate of the present invention is composed of a film 1 composed of a resin composition containing a fluorine-containing polymer and a support 2 as in Embodiment 2 of FIG. It is preferable that the oxygen gas permeability of the base material 10 combined with the film 1 is appropriately selected so as to be in the above range. As the support 2, it is particularly preferable to use a support that does not substantially interfere with the oxygen gas permeability of the film 1 such as a porous support or a mesh-shaped support.

  The method for forming the film is not particularly limited, and examples thereof include solution casting methods such as a solution casting method and a solution casting method; a calendar method; a press molding method. Among these methods, the solution casting method is preferable because of excellent productivity.

  As the solution for forming a film, the polymer solution or the polymer solution can be used. In one embodiment, a solution of the polymer may be preferred.

  As a typical method for forming a film, the resin (polymer) solution is applied to the surface of a film-forming support, for example, spin coating method, casting method, roll coating method, die coating method, gravure coating method, spray coating. Examples thereof include a method of forming a coating film by applying a usual method such as a method, a bar coating method, a flexographic printing method, or a dip coating method. The coating amount when the resin (polymer) solution is applied to the film-forming support is preferably such that the dry film thickness is 0.1 μm or more and 1 mm or less, and is 0.5 μm or more and 500 μm or less. It is more preferable to adjust so. Then, the film containing the said polymer can be obtained by removing a solvent and baking as needed.

Examples of the material constituting the film-forming support include quartz; inorganic glass such as glass, borosilicate glass, and soda glass; carbon; metal such as gold, silver, copper, silicon, nickel, titanium, aluminum, and tungsten; Polyolefins such as polyethylene and polypropylene; Polyesters such as polybutylene terephthalate (PBT) and polyethylene terephthalate (PET); Cyclic olefin resins such as cyclic olefin ring-opening polymerization / hydrogenated product (COP) and cyclic olefin copolymer (COC) Acrylic resin such as polymethyl methacrylate (PMMA); epoxy resin; AS resin (acrylonitrile-styrene copolymer), polyvinyl chloride, polyvinylidene chloride, polystyrene (PST), polystyrene resin, polyvinyl acetate, ABS resin , Polycarbonate Resin, vinyl ether, polyacetal (POM), polyamide, polyphenylene ether (PPE), polyaryl ether, polyphenylene sulfide (PPS), polysulfone (PS), polyethersulfone (PES), polyetheretherketone (PEEK), Resins such as polyaryletherketone (PEK), polyimide (PI), polyamic acid (PAA), polyamideimide acrylic resin, phenol resin, polyetherketone resin, polyethernitrile (PEN) resin; the above metals or oxides thereof Alternatively, glass having a mixed oxide or the like on its surface, metal, resin; wood and the like can be given. Examples of the mixed oxide include transparent conductive oxides such as ITO (indium tin oxide), SiO _{2 and the} like. Examples of the metal having a mixed oxide or the like on the surface include a SiO ₂ / Si base material. The film-forming support itself can be the “support” in Embodiment 2, and in this case, the cell culture substrate of the present invention is formed by a combination of the film and the support. In the second embodiment, the support can be in any form such as a plate or film, and may have the form of a cell culture container. Further, the film formed on the film-forming support may be peeled off after the film is formed, and the film alone may be used as the cell culture substrate of the first embodiment. Alternatively, the film peeled off from the film-forming support may be attached to and integrated with the surface of another support, and may be used as the cell culture substrate of Embodiment 2 including the film and the support. Arbitrary means, such as an adhesive agent, can be adopted as means for integrating the film and the support. The material and shape of the support in this case are the same as the material and shape of the film-forming support when the film-forming support is used as the support in Embodiment 2.

  Furthermore, the film of the resin composition may be stretched. The film may be stretched uniaxially or biaxially. Uniaxial stretching may be longitudinal stretching (stretching in the film winding direction) or lateral stretching (stretching in the film width direction). In the case of longitudinal stretching, it may be free end uniaxial stretching in which the change in the width direction of the film is free, or may be fixed end uniaxial stretching in which the change in the width direction of the film is fixed. Biaxial stretching may be sequential biaxial stretching in which transverse stretching is performed after longitudinal stretching, or simultaneous biaxial stretching in which longitudinal and transverse stretching are simultaneously performed. Further, stretching in the thickness direction of the film or stretching in the oblique direction with respect to the roll of the film may be performed. The stretching method, stretching temperature, and stretching ratio are preferably selected as appropriate according to the optical properties and mechanical strength of the target fluorine-containing polymer film.

  The total thickness (excluding the support) of the film composed of the resin composition containing the fluorine-containing polymer is preferably 0.1 μm or more and 1 mm or less, and preferably 0.5 μm or more and 500 μm or less. More preferably, it is 1 μm or more and 300 μm or less.

  In addition, it is preferable that the said resin solution heats the coating film of the said solution at the temperature and time which a solvent escapes, for example, in nitrogen atmosphere, Preferably it is 50-400 degreeC, More preferably, it is 100-300 degreeC, Preferably it is 10 It is possible to obtain a film composed of the resin composition by baking under conditions of minutes to 5 hours, more preferably 30 minutes to 3 hours.

  In the cell culture substrate of the present invention, the surface constituted by the resin composition containing the fluorine-containing polymer is preferably a smooth surface. As the smooth surface, for example, a surface having a surface roughness (centerline average roughness: Ra) of 0.5 μm or less is preferable. Preferably, it is 0.1 μm or less, more preferably 0.01 μm or less. In the present invention, the center line average roughness (Ra) is a value measured by a laser method, and can be measured using, for example, a surface roughness meter R5300GL-LA-A100-AC manufactured by Ryoka System. According to the present invention, cells can be cultured three-dimensionally on a smooth surface that can be easily produced. However, the surface constituted by the resin composition containing the polymer in the cell culture substrate of the present invention may be processed to have an appropriate roughness according to the purpose. For example, it is described in Non-Patent Document 1. It is also possible to form fine irregularities by rubbing treatment. In addition, a three-dimensional culture medium having a uniform size can be obtained by adding a cylindrical or conical hole (cavity) having a diameter of 50 to 500 μm and a depth of 50 to 500 μm (for example, 300 μm or less) to the cell culture substrate of the present invention For example, spheroids, three-dimensional cell aggregates, etc.) can be formed. Furthermore, by providing a cavity structure, it is possible to avoid removal of spheroids, three-dimensional cell aggregates, and the like from the base material together with the medium when the medium is removed.

  The cell culture substrate of the present invention preferably has the following effects in addition to the effects described so far. Since the base material of the present invention preferably has high heat resistance, high-pressure steam sterilization is possible. By performing high-pressure steam sterilization, there is no change in the quality of the substrate seen during γ-ray sterilization, and it is not necessary to remove residual gas during EOG sterilization. Risk and the risk of mixing components that inhibit the growth of cultured cells can be reduced. In addition, since a general polystyrene cell culture substrate has low heat resistance, high-pressure steam sterilization cannot be performed. In addition to the above sterilization method, the cell culture substrate of the present invention can be sterilized by a general sterilization method. Although it can sterilize by methods, such as EOG sterilization, these are examples and other sterilization methods may be adopted. Further, since the substrate of the present invention does not have autofluorescence near the excitation wavelength or fluorescence wavelength of fluorescent dyes generally used in immunostaining and the like, it can also be used for immunostaining using fluorescent dyes. Furthermore, the substrate of the present invention is preferably transparent and has a high oxygen permeability, and the oxygen supply to cells is improved by maintaining the dissolved oxygen concentration in the medium high, and the survival rate is high. And it becomes possible to culture | cultivate, maintaining the function of various cells highly. When the cell culture substrate of the present invention is a film-shaped cell culture substrate composed of the resin composition, the effects generally described in this paragraph are obtained.

3. Cell culture container The present invention also provides a cell culture container provided at least in part with the cell culture substrate having oxygen permeability. In a preferred embodiment, the cell culture container of the present invention has a cell culture substrate disposed such that one surface forms the bottom surface of the cell and medium containing portion and the other surface is exposed outside the container. Are provided at least in part.

  In one embodiment, the cell culture container of the present invention has a cell culture substrate as shown in FIG. 4-1, installed inside or at the bottom of the container, or one surface of the container for containing cells and medium. It may be provided with at least a part of a cell culture substrate disposed so that the bottom surface is formed and the other surface is exposed to the outside of the container.

  In the cell culture container of the present invention, the surface constituted by the resin composition containing the fluorine-containing polymer functions as a scaffold for cells to be cultured, so that the cell survival rate is high and the cell function is maintained high. Cell culture, particularly three-dimensional cell culture, is possible.

  The cell culture vessel of the present invention may be any shape as long as it includes the cell culture substrate of the present invention. For example, it can be in the form of various containers such as plates for culture such as single or multiwell plates, petri dishes, dishes, flasks, bags and the like. The cell culture container of the present invention may also be in the form of a cell culture container in a culture apparatus such as a mass culture apparatus or a perfusion culture apparatus.

  In another embodiment, in the cell culture container of the present invention, the surface of the cell culture substrate formed of the resin composition containing the fluorine-containing polymer of the substrate is a bottom surface of the cell and culture medium container. And the other surface of the base material is preferably disposed so as to be exposed to the outside of the container. That is, as shown in FIG. 1, when the cell culture substrate 10 is composed only of the film 1 made of a resin composition containing a fluorine-containing polymer, the cell culture substrate 10 has one of the main surfaces thereof. The bottom surface of the compartment containing the cells and the medium is formed, and the other main surface is disposed outside the container so as to be in contact with air or the like outside the container. As shown in FIG. 2, when the cell culture substrate 10 includes a film 1 and a support 2 made of a resin composition containing a fluorine-containing polymer, the cell culture substrate 10 is provided with the film 1. The surface S on the other side forms the bottom surface of the compartment containing the cells and the medium, and the surface on which the support 2 is disposed is arranged so as to be exposed to the outside of the container and to come into contact with air outside the container. In the cell culture container of the present invention, in the state of use for cell culture, the other surface of the cell culture substrate is exposed to the outside of the container and comes into contact with an oxygen-containing gas such as air outside the container. It is configured.

  In the cell culture vessel of the present invention of the above embodiment, the surface constituted by the resin composition containing a fluorine-containing polymer functions as a scaffold for cells to be cultured. And since the said base material has oxygen permeability, oxygen is supplied to a cell and a culture medium through the said base material from the surface exposed out of a container and contact | connecting oxygen-containing gas, such as air. This combination enables high cell viability and cell culture, particularly three-dimensional cell culture, while maintaining high cell function.

  The cell culture vessel of the present invention may be configured by combining the cell culture substrate of the present invention and other members, or the cell culture substrate of the present invention and other members are integrated. It may be comprised, and may be comprised only by the base material for cell cultures of this invention. When the cell culture substrate of the present invention is a flexible substrate such as a film, the bottom of the cell culture vessel is formed in a stretched state using an appropriate support member (such as a frame) having rigidity. It is also possible.

  FIG. 4A shows a cell culture container 100 which is an embodiment of the cell culture container of the present invention. A cell culture container 100 shown in FIG. 4A includes a wall member 20 that forms a container side wall and a container side wall erected from the edge of the container, and the cell culture substrate 10 is placed on the container bottom. It is formed. The configuration of the cell culture substrate 10 is as described above. The inner shape and the outer shape when the wall member 20 is viewed in plan from the opening side can be any shape such as, for example, a circle or a polygon (square, triangle, etc.).

  FIG. 4-2 (a) shows a cell culture container 100 which is an embodiment of the cell culture container of the present invention. A cell culture container 100 shown in FIG. 4-2 (a) includes a cell culture substrate 10 that forms the bottom of the container, and a wall member 20 that forms a container side wall that stands up from the edge of the cell culture substrate 10. The cell 101 is formed by the cell culture substrate 10 and the wall member 20. The structure of the cell culture substrate 10 is as described above, and the surface S constituted by the resin composition containing the fluorine-containing polymer is disposed so as to face the inside of the container (the accommodating part 101). The inner shape and the outer shape when the wall member 20 is viewed in plan from the opening side can be any shape such as, for example, a circle or a polygon (square, triangle, etc.).

  A cell culture container 100 shown in FIGS. 5 (a), 5 (b) and 5 (c) is a multiwell plate which is another embodiment of the cell culture container of the present invention. A cell culture container 100 shown in FIGS. 5 (a), 5 (b) and 5 (c) is composed of a cell culture substrate 10 and a resin composition containing the fluorine-containing polymer of the cell culture substrate 10. And a plate-like wall member 20 formed with a plurality of (24 in the figure) through-holes penetrating in the thickness direction and disposed so as to cover the surface S. A plurality of accommodating portions 101 for accommodating cells and culture media are formed by the portion surrounding each through-hole of the wall member 20 and the cell culture substrate 10. The structure of the cell culture substrate 10 is as described above, and the surface S constituted by the resin composition containing the fluorine-containing polymer is disposed so as to face the inside of the container (the accommodating part 101). Here, in FIG.5 (c), the surface S comprised by the resin composition containing a fluorine-containing polymer faces the inside of a container (accommodating part 101), and the surface on the opposite side to the surface S of the base material 10 for cell cultures However, when the cell culture vessel 100 is placed on a flat surface, the wall member 20 does not come into contact with the flat surface, and a gap 4 is formed between the surface opposite to the surface S and the flat surface. It is connected to the.

  A cell culture container 100 shown in FIG. 6 is a further embodiment of the cell culture container of the present invention. A cell culture container 100 shown in FIG. 6 includes a cell culture base material 10 that forms the bottom of the container and a wall member 20 that forms the side wall of the container. 101 is formed. Here, the cell culture substrate 10 has a surface S composed of a resin composition containing a fluorine-containing polymer facing the inside of the container (accommodating portion 101), and is opposite to the surface S of the cell culture substrate 10. When the cell culture container 100 is placed on a flat surface, the wall member does not come into contact with the flat surface, and a gap 4 is formed between the surface opposite to the surface S and the flat surface. 20 is connected.

  4-2 (a), FIGS. 5 (a), 5 (b) and 5 (c), and the cell culture container 100 of each embodiment described in FIG. The cell culture substrate 10 may be connected by any means, for example, it can be connected via an adhesive material such as a pressure-sensitive double-sided tape or an adhesive member.

  As described above, a cell culture container having a cell culture substrate having a surface S composed of a resin composition containing a fluorine-containing polymer disposed on the bottom surface of the container and the other surface exposed outside the container is manufactured. be able to. The cell culture vessel of the present invention is not limited to this form, and can be in any form.

4). Culturing method The present invention also provides a method for culturing cells, the method comprising culturing cells on the surface of the cell culture substrate comprising a resin composition containing the fluorine-containing polymer. To do.

  Three-dimensional culture is performed by culturing cells capable of forming a three-dimensional tissue for an appropriate time on the surface of the cell culture substrate of the present invention, which is composed of the resin composition containing the fluorine-containing polymer. be able to. However, the cell culture method of the present invention is not necessarily limited to such a form. A form in which cells that do not form a three-dimensional tissue are cultured, or a cell that can form a three-dimensional tissue is transformed into a three-dimensional tissue. The form etc. which culture | cultivate to the stage before forming are included.

  Specifically, as shown in FIG. 4-1, the cell culture substrate 10 (having the structure shown in FIG. 1 or FIG. 2) is placed in a container, and is constituted by a resin composition containing a fluorine-containing polymer. The cells may be cultured in a state where the cells and the medium are in contact with the surface S (see FIGS. 1 and 2).

  In another embodiment, the present invention is also a method for culturing cells, wherein the cell culture medium is in contact with a surface constituted by a resin composition containing a fluorine-containing polymer of the cell culture substrate, A method comprising culturing cells in a state where the other surface of the substrate for use is in contact with an oxygen-containing gas such as air is provided.

  Specifically, as shown in FIG. 3, the surface S (FIGS. 1 and 2) of the cell culture substrate 10 (having the structure shown in FIG. 1 or 2) composed of a resin composition containing a fluorine-containing polymer. The cells may be cultured in a state where the cells 3 and the culture medium 2 are in contact with each other and the other surface of the cell culture substrate 10 is in contact with an oxygen-containing gas such as air. In this method, the surface S composed of the resin composition containing the fluorine-containing polymer of the base material 10 functions as a scaffold for cells to be cultured. And since the base material 10 has oxygen permeability, oxygen is supplied to the cell 3 and the culture medium 2 through the base material 10 from the surface which contact | connects oxygen-containing gas, such as air. According to this method, it is possible to perform cell culture, particularly three-dimensional cell culture, with high cell viability and maintaining high cell function. For example, when the above-described cell culture container 100 shown in FIG. 4-2 (a) is used, at least a part of the lower surface of the cell culture container 100 (the surface opposite to the surface S of the cell culture substrate 10) is The cell culturing method described above can be realized by arranging the cell culturing container 100 so as to be in contact with an oxygen-containing gas such as air and culturing the cell. For example, as shown in FIG. 4B, if a suitable spacer 200 having a small width is arranged on the flat surface 300 and the cell culture vessel 100 is placed on the spacer 200, the vessel of the culture substrate 10 is obtained. The exposed surface can be in contact with an oxygen-containing gas (air). The cell culture can be performed using the cell culture vessel 100 arranged in this way. Even when the cell culture vessel 100 is placed directly on the flat surface 300 without using the spacer 200, the surface of the culture substrate 10 exposed to the outside of the container and the flat surface 300 are usually partially. Can contain the oxygen-containing gas (air), and the cell culture method of the present invention can be performed. The cell culture container 100 shown in FIGS. 5A, 5B, and 5C can be used similarly. When the cell culture container 100 shown in FIG. 5C and FIG. 6 is placed on a flat surface, the bottom end of the wall member 20 protrudes below the cell culture substrate 10. The end of the wall member 20 functions as a spacer, and a space 4 is formed between the surface opposite to the surface S of the cell culture substrate 10 and a flat surface, and an oxygen-containing gas is formed in the space 4. Since (air etc.) can exist, the cell culture method of this invention can be performed easily.

  The types of cells cultured by the cell culture method of the present invention are not particularly limited. For example, human normal hepatocytes, rat normal hepatocytes, mouse normal hepatocytes, human liver cancer cells, human hepatoblastoma cells, rat hepatoma Cells, mouse hepatoma cells, induced pluripotent stem cells (iPS cells), embryonic stem cells (embryonic stem cells: ES cells), mesenchymal stem cells, etc. can generally be three-dimensionally cultured Adipose cells, hepatocytes, kidney cells, pancreatic cells, mammary cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, cardiomyocytes, nerve cells, including the required cells and various progenitor cells and stem cells, Glial cells, dendritic cells, chondrocytes, osteoblasts, osteoclasts, bone cells, fibroblasts, various blood cells, other mesenchymal precursors Other cells such as cells and various cancer cells can be mentioned.

  The cells can be cultured in a suitable medium. The type of medium is not particularly limited. For example, any cell culture basic medium, differentiation medium, primary culture medium, or the like can be used. Specific examples include Dulbecco's modified Eagle medium (DMEM), Glasgow MEM (GMEM), RPMI 1640, Ham F12, MCDB medium, Williams medium E, etc., but are not limited thereto, and cells are required for growth and differentiation. Any medium containing components can be used. Furthermore, a medium supplemented with serum, various growth factors, or differentiation-inducing factors may be used.

5). Three-dimensional culture The present invention is also a method for three-dimensional culture of cells, the step of three-dimensionally culturing cells on the surface of the cell culture substrate comprising the resin composition containing the fluorine-containing polymer. A method comprising: That is, using the cell culture method of the present invention, cells can be three-dimensionally cultured to form a tissue.

  Here, examples of the tissue formed by three-dimensional culture include spheroids and three-dimensional cell aggregates. Spheroids or three-dimensional cell aggregates are spheroids or three-dimensional cell aggregates formed of a single cell such as a normal rat hepatocyte, such as various fibroblasts and vascular endothelial cells and 2 normal rat hepatocytes. It may be a spheroid or a three-dimensional cell aggregate in which different cell types are mixed. Examples of cells that can be used include the various cells described above.

  The type of medium used for the three-dimensional culture is not particularly limited. For example, any cell culture basic medium, differentiation medium, primary culture medium, or the like can be used. Specific examples include Dulbecco's modified Eagle medium (DMEM), Glasgow MEM (GMEM), RPMI 1640, Ham F12, MCDB medium, Williams medium E, etc., but are not limited thereto, and cells are required for growth and differentiation. Any medium containing components can be used. Furthermore, a medium supplemented with serum, various growth factors, or differentiation-inducing factors may be used.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention. In the following, “part” means “part by mass” and “%” means “mass%” unless otherwise specified.

<Measurement of water contact angle>
Apparatus: Automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500)
Measurement method: The adhesion angle of the droplet immediately after the dropwise addition of 2 μl of water at a temperature of 25 ° C. was measured.

<Dynamic viscoelasticity measuring method>
Apparatus: Dynamic Viscoelasticity RSA III manufactured by TA Instruments
Measurement method: A film having a thickness of 60 μm was produced in a strip shape of 5 × 40 mm, the elongation and stress at 25 ° C. were measured, and the tensile modulus was calculated.

<Measurement of oxygen gas permeability and oxygen gas permeability coefficient>
The oxygen gas permeability (unit: cm ³ (STP) / (m ² · 24 h · atm)) and the oxygen gas permeability coefficient (unit: cm ³ (STP) · cm / (cm ² · s · cmHg)) are JIS K7126. -1 (Differential pressure method) Measured by a method according to Appendix 2. Specifically, the test was performed under the following conditions.
Test method: Differential pressure method (according to JIS K7126-1 Annex 2)
Detector: Gas chromatograph (Thermal conductivity detector: TCD)
Test differential pressure: 1 atm
Test gas: Oxygen gas (dry state (relative humidity is almost 0%))
Test conditions: 25 ° C ± 2 ° C
Transmission area: 1.52 × 10 ⁻³ m ²
Equipment: Differential pressure type gas / vapor permeability measuring device (GTR-30XAD2, G2700T · F)
GTR Tech Co., Ltd./Yanaco Technical Science Co., Ltd.

<< Preparation Example 1 >> Fluorine-containing polyaryletherketone resin (FPEK)
In a 225 ml three-necked flask, 16.74 g of 4,4′-bis (2,3,4,5,6-pentafluorobenzoyl) diphenyl ether (abbreviation p, p-BPDE), 2,2-bis (4 - were charged hydroxyphenyl) hexafluoropropane (6FBA) 10.14g, potassium carbonate _{(K 2} CO 3) 4.14g and N- methylpyrrolidinone 90 g. The mixture was heated to 60 ° C. and heated for 5 hours. After completion of the reaction, the mixture was cooled, and this solution was poured into a 1% aqueous acetic acid solution with vigorous stirring using a blender. The precipitated polymer was separated by filtration, washed with distilled water and methanol, and then dried under reduced pressure.

The obtained powdery fluorinated polyaryletherketone was dissolved in a 2-butanone solution to a concentration of 15% to obtain a fluorinated polyaryletherketone (FPEK) solution having the following structure.

<< Example 1 >> FPEK film The FPEK solution obtained in Preparation Example 1 was formed into a film on a glass substrate using a die coater so that the thickness of the polymer film after firing was 60 μm. After heating at 150 ° C. for 1 hour and firing, the film was peeled off from the glass to obtain a fluorine-containing polyaryletherketone film (FPEK film).

The obtained FPEK film has a thickness of 60 μm, the contact angle of water is 89 °, the tensile elastic modulus is 1.2 GPa, and the oxygen transmission coefficient is 3.21 × 10 ⁻¹⁰ cm ³ (STP) cm / (cm ² · s · cmHg).

Example 2 Spheroid Formation by Cell Culture
1. Acquisition of rat primary hepatocytes A Wistar rat, male, 9 weeks old, 200 g body weight of a specific viral pathogen free was purchased from SLC Japan. The rat primary hepatocytes were obtained by referring to the method described in Chapter 10 of the Cultured Cell Experiment Handbook (Yodosha), hepatocytes. Specifically, Wistar rats were laparotomized under isoflurane anesthesia, a catheter was inserted into the portal vein, and a preperfusion solution having the composition described below was injected. At the same time, the lower vena cava under the liver was incised to release blood. Next, the chest cavity was opened, the inferior vena cava entering the right atrium was incised, and the inferior vena cava below the liver was stopped and perfusion was performed. After confirming that blood removal from the liver was sufficiently performed, the perfusion was stopped, and the perfusion solution was replaced with a collagenase solution having the composition shown in Table 1 below. After confirming that the intercellular tissue was digested by collagenase, the perfusion was stopped. After the liver was cut off and transferred to a glass petri dish, a cold Hanks solution was added and the cells were dispersed by pipetting. Next, undigested tissue was removed with a 150 mm filter. The cell suspension was centrifuged at 50G for 1 minute several times to remove non-parenchymal cells. The survival rate of the obtained hepatocytes was measured by trypan blue exclusion method, and hepatocytes with a survival rate of 70% or more were used as rat primary hepatocytes in the culture test.

2. Culture of rat primary hepatocytes Rat primary hepatocytes obtained by the above-described method are suspended in a medium having the following composition and 1.25 × 10 ⁵ cells / mL so as to be 2.66 × 10 ⁴ cells / cm ^2. The rat primary hepatocyte suspension (0.4 mL) was added to a collagen type I-coated microplate 24 Well with lid (Asahi Glass Co., Ltd.) and an FPEK membrane, and cultured under conditions of 37 ° C. and 5% CO ₂ . The FPEK membrane prepared in Example 1 was used for cell culture after autoclaving. The medium was exchanged 3 hours after seeding, on the 1st, 3rd, and 5th days of culture.

Medium composition William's E medium (Wako Pure Chemicals) + 10% FBS (Wako Pure Chemicals) + 8.6 nM insulin + 255 nM dexamethasone + 50 ng / mL EGF + 5 KIU / mL aprotinin + antibiotics (penicillin (100 units / mL) / streptomycin (100 μg / mL) / Amphotericin B (0.25 μg / mL))

  Phase contrast micrographs of the collagen type I-coated microplate 24Well (Asahi Glass Co., Ltd.) and the FPEK film on the fifth day of culture are shown in FIGS. 7A and 7B, respectively.

  In the collagen type I coated microplate 24Well (Asahi Glass Co., Ltd.) with a lid generally used for culturing adherent cells, the cells adhered to the substrate in a single layer and almost no formation of cell aggregates was observed, but the FPEK membrane Then, cells attached in a monolayer were not observed, and cell aggregates having a three-dimensional structure were formed. The size of the cell agglomerates was uniform and appropriate, and spheroids were uniformly distributed over the entire surface of the substrate.

  After treatment with a 0.25% trypsin / 50 mM EDTA solution every 24 hours of culture, the total cell number was measured using a 0.4 w / v% trypan blue solution (Wako Pure Chemical Industries) and a hemocytometer. Further, the culture solution was sampled every 24 hours and stored at -20 ° C.

3. Albumin quantification was performed using the culture solution of each test section on the fifth day of quantification of albumin culture. Rat Albumin ELISA Quantitation Set (Bethyl Laboratories) was used for quantification of albumin, and albumin quantification experiments were performed according to the attached protocol. The results of albumin quantification in each test group are shown in FIG.

  As shown in FIG. 8, it was confirmed that the FPEK membrane produced a larger amount of albumin than the collagen type I-coated microplate 24 Well (Asahi Glass Co., Ltd.) with a lid. It is considered that high liver function was expressed in cell aggregates of appropriate size formed on the FPEK membrane.

Claims (7)

A cell culture substrate, wherein at least a part of the surface is composed of a resin composition containing a fluorine-containing polymer,
The fluorine-containing polymer has the following formula (II-1):

[Wherein R ⁴² has the following structure:

One of them. ]
A cell culture substrate comprising a fluorine-containing aryl ether ketone polymer containing a repeating unit represented by the formula:   The cell culture substrate according to claim 1, wherein the fluorine content in the resin composition is 1 to 60 mass%. A cell culture container,
A cell culture container, wherein at least a part of the container is composed of the cell culture substrate according to claim 1 or 2 . A method for culturing cells comprising:
A method comprising culturing cells on the surface of the cell culture substrate according to claim 1 or 2 constituted by the resin composition. A method for culturing cells comprising:
Using a base material in which at least a part of one surface is composed of a resin composition containing a fluorine-containing polymer, cells and a medium are in contact with the one surface of the base material, and the other surface of the base material is oxygen Including a step of culturing the cells in contact with the contained gas,
The fluorine-containing polymer has the following formula (II-1):

[Wherein R ⁴² has the following structure:

One of them. ]
A fluorine-containing aryl ether ketone polymer containing a repeating unit represented by the formula : The method according to claim 4 or 5 , wherein the culturing step is a step of culturing cells three-dimensionally. The method according to claim 6 , wherein the step of three-dimensionally culturing cells is a step of culturing the cells to form spheroids or three-dimensional cell aggregates.

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