JP2017163898A - Method of culturing neural stem cell, and method of forming neurospheroid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique which enables the formation of neurospheres without the need for suspension culture of neural stem cells.SOLUTION: A method of culturing neural stem cells comprises culturing cells comprising neural stem cells on a fluorine-containing polymer base material, and forming a neurosphere held on the base material.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for culturing neural stem cells and a method for forming neurospheroids.

  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. It is done. In the case of culturing cells on the surface of a general resin cell culture substrate, it is difficult to form a three-dimensional network by spreading the cells in a plane and proliferating (two-dimensional culture).

  Neural stem cells are cells that have the ability to differentiate into nerve cells or glial cells (for example, astrocytes, oligodendrocytes, etc.) and retain the proliferation ability (self-replication ability). By culturing neural stem cells in suspension, it is possible to form a three-dimensional network singly or with other cells to form cell aggregates called neurospheres. In the case of neural stem cells, when culturing is performed on the surface of a general resinous cell culture substrate, differentiation is induced by adhesion to the substrate and proceeds. For this reason, it is necessary to avoid adhesion with the base material in order to grow and maintain neural stem cells in an undifferentiated state to form neurospheres. Conventionally, formation of neurospheres has been performed by suspension culture. (Conventional “neurosphere method”). By inducing differentiation of neural stem cells using the formed neurospheres, it is also possible to form neurospheroids that are cell aggregates containing neurons and glial cells. Neurospheres containing undifferentiated and proliferating neural stem cells, and neurospheroids that have a three-dimensional network similar to living organisms are used to study and treat neurological diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. It is a very useful tool in law development.

  For the above reasons, in the development of cell culture vessels used for the formation of neurospheres, contrivances have been made to reduce the adhesion of neural stem cells to the culture vessel surface. For example, as a technique for reducing the adhesion of neural stem cells to the surface of a culture container, Patent Document 1 discloses a method for producing a neural stem cell aggregate formation container, wherein a water-soluble resin is coated on the inner surface of the container to form a water-soluble material. A water-soluble resin coating step for forming a resin coating layer, and a water-insoluble cured film modification step for curing the water-soluble resin coating layer and modifying it to a water-insoluble cured film layer after the step. An invention relating to a method for producing a neural stem cell aggregate formation container is disclosed.

  On the other hand, after differentiation induction, there is no need to proliferate neural stem cells in an undifferentiated state, and there is no need to cultivate in a floating system. Therefore, from the viewpoint of operability, the culture of neurospheroids is generally performed in a state of being fixed on a substrate. For this reason, when forming neurospheroids, after culturing neural stem cells in a culture vessel with low cell adhesion as described above, transfer the formed neurospheres to a culture vessel with high cell adhesion. A technique of inducing differentiation has been taken. However, performing differentiation induction using a culture vessel different from the culture vessel used when forming the neurosphere not only complicates the work process, but also the formed neurosphere collapses when the medium is replaced, There was also some loss with it. In order to solve such a problem, as a technique capable of forming a neurosphere and a neurospheroid in a single culture vessel, for example, in Patent Document 2, a group having a predetermined chemical structure exists in the vicinity of the surface. Using a culture container to form a cell aggregate by suspension culture of neural stem cells in a medium containing a growth factor, and adding a differentiation-inducing factor to the medium to adhere the cell aggregate to the culture container And an invention relating to a culture method for forming neural cells and / or glial cells from neural stem cells, comprising the step of differentiating said neural stem cells.

JP 2008-220205 A JP 2013-135640 A

  However, in the culture method described in Patent Document 2, it is possible to perform neurosphere formation from neural stem cells to differentiated neurons and / or glial cells using a single culture vessel, but the formation of neurospheres. Must be done in suspension culture. Cultures in suspension systems still have problems such as neurosphere collapse and loss. In addition, when neurospheres are cultured in a floating system, excessively large neurospheres are formed due to the association of the neurospheres floating in the medium, and the cells existing in the central part of the neurospheres may be necrotic. Furthermore, when differentiation induction is performed on neural stem cells, it is necessary to collect the formed neurospheres once and then perform differentiation induction processing, which makes handling complicated.

  Therefore, the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a technique capable of forming neurospheres without requiring floating culture of neural stem cells.

  Another object of the present invention is to provide a technique capable of forming neurospheres and forming neuropheroids in a single cell culture vessel.

  The present inventors have conducted intensive research to solve the above problems. As a result, it has been found that the above-mentioned problems can be solved by culturing neural stem cells on a fluoropolymer substrate, and the present invention has been completed.

  According to the present invention, it is possible to provide a technique capable of forming neurospheres without requiring suspension culture of neural stem cells.

  According to the present invention, it is also possible to provide a technique capable of forming neurospheres and neurospheroids in a single cell culture vessel.

FIG. 1 is an optical microscope image obtained by culturing neural stem cells on the fluoropolymer substrate in Example 1 and confirming that neurospheres retained on the substrate are formed (FIG. 1 (a) culture). 1st day, (b) 3rd culture day, (c) 5th culture day, (d) 8th culture day). In FIG. 1, the upper part of each of (a) to (d) is 40 times magnification, the lower part is 200 times, and the white bar is 200 μm. FIG. 2 is an optical microscopic image obtained by performing differentiation induction treatment on neural stem cells cultured on the fluoropolymer substrate in Example 1 and confirming that neuropheroids are formed (FIG. 2 (a) differentiation medium). On the first day of the medium replacement, the first day of culture in differentiation medium, (b) the third day of culture in differentiation medium, (c) the fifth day of culture in differentiation medium, (d) in the differentiation medium Culture day 9). In FIG. 2, the upper part of each of (a) to (d) is 40 times magnification, the lower part is 200 times, and the white bar is 200 μm. FIG. 3 is an optical microscope image of the neurospheroid formed by suspension culture in Comparative Example 1 (FIG. 3 (a) culture day 1, (b) culture day 3, (c) culture day 5; d) Day 8 of culture). In FIG. 3, the upper part of each of (a) to (d) is 40 times magnification, the lower part is 200 times, and the white bar is 200 μm. FIG. 4 is an optical microscopic image in the case where differentiation induction treatment is performed on the neurospheroid formed in suspension culture in Comparative Example 1 (FIG. 4 (a), the day of medium exchange to the differentiation medium is regarded as day 0, Day 1 of culture in differentiation medium, (b) Day 3 of culture in differentiation medium, (c) Day 5 of culture in differentiation medium, (d) Day 9 of culture in differentiation medium). In FIG. 4, the upper part of each of (a) to (d) is 40 times magnification, the lower part is 200 times, and the white bar is 200 μm. FIG. 5 shows the results of measuring the expression levels of undifferentiated markers and differentiation markers expressed in cells before and after differentiation induction in Example 1 and Comparative Example 1, respectively.

  One aspect of the present invention relates to a method for culturing neural stem cells, comprising culturing cells containing neural stem cells on a fluoropolymer substrate and forming neurospheres retained on the substrate. The present invention is based on the surprising finding that by culturing neural stem cells on a fluorine-containing polymer substrate, the neural stem cells can be grown in an undifferentiated state while being retained on the substrate, and neurospheres can be formed. Is. According to the present invention, it is not necessary to form neurospheres by suspension culture, so that the risk that the formed neurospheres collapse during medium exchange or partly lose with the medium is greatly reduced. Can do. In addition, according to the present invention, it is possible to effectively prevent an excessively large neurosphere from being formed due to the association of neurospheres floating in the medium. The detailed mechanism by which neurospheres that could not be formed on the substrate in the past can be formed by culturing neural stem cells on a fluoropolymer substrate is unknown. The reason is guessed. That is, although the detailed mechanism by which differentiation induction proceeds by adhesion of neural stem cells to the substrate is unclear, physical stimulation of cell contact with the substrate is involved as an input for the differentiation induction signal. it is conceivable that. On the other hand, fluoropolymers are characterized by being hydrophobic and having high water repellency. Therefore, by culturing neural stem cells on a fluoropolymer substrate, the hydrophobic nature of the fluoropolymer makes the interaction between the substrate and neural stem cells appropriate, and the differentiation induction signal is suppressed. It is presumed that the neurospheres can be retained on the material. In addition, the said mechanism is estimation and does not restrict | limit the technical scope of this invention.

  Another aspect of the present invention is that the neural stem cells are cultured on a fluoropolymer substrate to form a neurosphere held on the substrate, and the neurosphere is held on the substrate. Inducing differentiation of a stem cell, the present invention relates to a method for forming a neurospheroid. Such a technique capable of inducing differentiation of a neurosphere held on a substrate enables formation of neurosphere and formation of neuropheroid in a single cell culture vessel. In addition, although the detailed mechanism is unknown, the present inventors performed induction treatment using an inducer on neurospheres retained on a fluoropolymer, and the cells became highly sensitive to the inducer. It has also been found that there is an advantage in responding that differentiation is strongly induced.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment.

  In this specification, “X to Y” indicating a range means “X or more and Y or less”. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50% RH.

<Neural stem cell culture method>
In the present invention, neural stem cells are cultured on a fluoropolymer substrate. Examples of the fluorine-containing polymer include fluorine-containing polyimide, fluorinated polyaryletherketone, fluorinated polyester polyether, and fluorinated polyether. The fluorine-containing polymer forming the substrate may be a blend of two or more types of polymers having different chemical structures. As a fluorine-containing polymer, it is preferable to contain an aromatic fluorine compound as a structural unit from a viewpoint of the intensity | strength and heat resistance of the film obtained. In one embodiment, the fluorine-containing polymer is preferably a fluorine-containing polyimide, more preferably a fluorine-containing polyimide containing a structural unit represented by the following formula (II).

(Fluorine-containing polyimide)
The fluorine-containing polyimide used in the present invention is a polyimide having one or more fluorine atoms in a repeating unit. Typically, a polyamic acid obtained by polymerizing at least one acid dianhydride and one or more diamines is used. It is a fluorine-containing polyimide obtained by imidization. In the present invention, the fluorine-containing polyimide constituting the surface serving as a scaffold for cells may contain polyamic acid as part of its chemical structure. In order to produce a fluorine-containing polyimide, a compound having a fluorine atom in the molecule is used as at least one compound of acid dianhydride and diamine.

  As a method for producing polyimide, a two-step synthesis method or a one-step synthesis method can be used.

  The two-step synthesis method of polyimide is a method of synthesizing polyamic acid as a precursor and converting the polyamic acid into polyimide acid. The polyamic acid as the precursor may be a polyamic acid derivative. Examples of the polyamic acid derivatives include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amides, polyamic acid derivatives from bismethylidene pyromellitide, polyamic acid silyl esters, and polyamic acid isoimides.

  As a one-step synthesis method of polyimide, for example, a one-step synthesis method using a solvent such as a high temperature melt polymerization method, an isocyanate method, a tetracarboxylic acid dithioanhydride method, or a method using an ionic liquid can be used. Other one-step synthesis methods include a polymerization method via a nylon salt monomer, a high temperature solid phase polymerization method, a high temperature solid phase polymerization method under high pressure, and a solid phase polymerization method in water.

  In the present invention, the “acid dianhydride residue” may be a tetravalent organic group that forms the above structure, and need not actually be a residue formed by reaction of acid dianhydride. Similarly, the “diamine compound residue” may be a divalent organic group that forms the above structure, and does not need to be a residue actually formed by reaction of a diamine compound.

  The fluorine-containing polyimide has the following formula (I) in the main chain:

[In formula (I),
X ⁰ is a tetravalent organic group, Y ⁰ is a divalent organic group,
The total number of fluorine atoms contained in X ⁰ and Y ⁰ is one or more,
Y ⁰ is a structure of a diamine compound containing a biphenyl group, wherein each of the two benzene rings of the biphenyl group is substituted with one amino group, wherein each amino group is substituted with a single bond to a nitrogen atom. Has a structure. ]
The repeating unit shown by is included.

  In this invention, the fluorine-containing polyimide containing the structural unit shown by the following formula | equation (II) is preferable from a viewpoint of neurosphere formation property.

In the formula (II), X represents an oxygen atom, a sulfur atom, or a divalent organic group; Y represents a divalent organic group; Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and X, Y, Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and At least one of Z ⁶ contains one or more fluorine atoms, and p is 0 or 1. In the fluorine-containing polyimide, the chemical structure represented by the formula (II) may be different for each structural unit of the polymer, or may be the same.

  In the above formula (II), when p = 0, X may not be present (in other words, the left and right benzene rings may be directly bonded), but when p = 1 , The left and right benzene rings are bonded via X.

  Specific examples of the divalent organic group represented by X include an alkylene group, an arylene group, an aryleneoxy group, an arylenethio group, and the like. Among these, an alkylene group, an aryleneoxy group, and an arylenethio group are exemplified. Preferably, an alkylene group and an aryleneoxy group are more preferable, and these may be substituted with a fluorine atom, and it is preferable that all the hydrogen atoms in the divalent organic group are substituted with a fluorine atom. Carbon number of the said alkylene group is 1-12, for example, Preferably it is 1-6.

The alkylene group substituted with a fluorine atom are examples of X, for _{example, -C (CF 3) 2 -} , - C (CF 3) 2 -C (CF 3) 2 - and the like can be exemplified. Among the above-described alkylene groups as examples of X, —C (CF ₃ ) ₂ — is preferable.

  As an arylene group which is an example of X, the following can be illustrated, for example.

  As an aryleneoxy group which is an example of X, the following can be illustrated, for example.

  As an arylenethio group which is an example of X, the following can be illustrated, for example.

  The above-mentioned arylene group, aryleneoxy group and arylenethio group which are examples of X are each independently a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom or a chlorine atom) And more preferably a fluorine atom.), And a group selected from the group consisting of a methyl group and a trifluoromethyl group. There may be a plurality of these substituents, in which case the types of the substituents may be the same or different. Suitable substituents substituted on the arylene group, aryleneoxy group and arylenethio group are a fluorine atom and / or a trifluoromethyl group, and preferably a fluorine atom. The arylene group, aryleneoxy group and arylenethio group are preferably substituted with at least one fluorine atom when Y does not contain a fluorine atom.

  In the above formula (II), the divalent organic group represented by Y is not particularly limited, and examples thereof include a divalent organic group having an aromatic ring. Specifically, a group consisting of one benzene ring or two or more benzene rings are a single bond, an alkylene group having 1 to 4 carbon atoms (for example, a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, An isobutylene group), an oxygen atom, a sulfur atom or a group having a structure bonded directly. Preferably, the divalent organic group represented by Y is a structure in which two or three benzene rings each independently having a substituent are bonded via a single bond, an oxygen atom, or a sulfur atom. It is group which has. More preferably, the divalent organic group represented by Y has a structure in which two or three benzene rings each independently having a substituent are bonded via an oxygen atom or a sulfur atom. It is a group. Examples of the substituent on the benzene ring include a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group and trifluoro. Examples include groups selected from the group consisting of methyl groups.

  Specific examples of the divalent organic group represented by Y include the following groups.

  The divalent organic group having an aromatic ring described above as an example of Y is a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom or a chlorine atom, if it can be substituted). , More preferably a fluorine atom.), Which may be substituted with a group selected from the group consisting of a methyl group and a trifluoromethyl group. There may be a plurality of these substituents, in which case the types of the substituents may be the same or different. A preferred substituent substituted with a divalent organic group having an aromatic ring is preferably a fluorine atom and / or a trifluoromethyl group, particularly when X does not contain a fluorine atom.

  Y may be a structure represented by the following formula (III).

In the formula (III), B ¹ represents CF ₃ or CN. B ² is the same or different and represents H, F, Cl, Br or I. R ¹ represents a halogen-substituted alkyl group having 1 to 20 carbon atoms. X ^A is the same or different and represents O or S. X ^B represents O or S. n represents the number of substituents of the groups represented by ^{B 2,} is an integer of 0-2. m ^represents the number of substituents of the group represented by X B ^{R 1,} is an integer of 1 to 3. Further, n + m = 3.

In the formula (III), R ¹ represents a halogen-substituted alkyl group, and the halogen-substituted alkyl group means a group in which at least a part of hydrogen atoms bonded to carbon atoms constituting the alkyl group is substituted with a halogen atom. The structure is not particularly limited, and may be any structure of a linear, branched, or cyclic alkyl group. Further, the halogen-substituted alkyl group may have an ether bond. The halogen atom is preferably a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br) or an iodine atom (I), and may be substituted with two or more of these atoms. R ¹ is preferably a fluorine-substituted alkyl group having 1 to 20 carbon atoms. R ¹ preferably has 2 to 18 carbon atoms, and more preferably 3 to 15 carbon atoms.

Examples of the group particularly suitable as R ¹ include at least one group selected from the group represented by the following chemical formula.
_{CF 3 - (CF 2) 7} - (CH 2) 2 -
_{CF 3 - (CF 2) 9} - (CH 2) 2 -
_{CF 3 - (CF 2) 2} -CH 2 -
CF ₃ — (CF ₂ ) ₃ —CH ₂ —
_{CHF 2 - (CF 2) 7} -CH 2 -
(CF ₃ ) ₂ -CF (CF ₂ ) _2- (CH ₂ ) _2-
CF ₃ CH ₂ −
HCF ₂ CH ₂ −
F (CF ₂ ) ₂ CH ₂ —
CHF ₂ CF ₂ CH _2-
(CF ₃ ) ₂ CH—
CF ₃ CH ₂ CH _2-
H (CF ₂ ) ₂ CH ₂ —
_{Cl (CF 2) 2 CH 2} -
(CF ₃ ) C (CH ₃ ) H—
_{F (CF 2) 3 CH 2} -
F (CF ₂ ) ₂ (CH ₂ ) ₂ −
CF ₃ CHFCF ₂ CH _2-
CF ₃ (CH ₂ ) ₃ −
_{F (CF 2) 2 C (} CH 3) H-
CF ₃ C (CH ₃ ) ₂ −
_{CH 3 C (CF 3) 2} -
(CF ₃ ) ₄ C-
(CF ₃ ) ₂ C (CCl ₃ ) −
F (CF ₂ ) ₄ CH _2-
F (CF ₂ ) ₃ (CH ₂ ) ₂ −
F (CF ₂ ) ₂ (CH ₂ ) ₃ −
CF ₃ (CH ₂ ) ₄ −
(CF ₃ ) ₂ CFCH ₂ CH _2-
(CF ₃ ) ₂ C (CH ₃ ) CH ₂ —
_{H (CF 2) 4 CH 2} -
_{Cl (CF 2) 4 CH 2} -
Br (CF ₂ ) ₂ (CH ₂ ) ₃ −
_{CF 3 CH 2 CH (CH 3} ) CH 2 -
_{CF 3 CF (OCF 3) CH} 2 CH 2 -
(CF ₃ ) ₂ CHOCH ₂ CH ₂ —
_{F (CF 2) 3 C (} CH 3) H-
F (CF ₂ ) ₅ CH ₂ —
F (CF ₂ ) ₄ (CH ₂ ) ₂ −
F (CF ₂ ) ₃ (CH ₂ ) ₃ −
F (CF ₂ ) ₂ (CH ₂ ) ₄ −
(CF ₃ ) ₂ CF (CH ₂ ) ₃ −
(CF ₃ ) ₃ CCH ₂ CH _2-
CF ₃ CF (OCF ₃ ) (CH ₂ ) ₃ −
_{F (CF 2) 3 OCF (} CF 3) CH 2 -
H (CF ₂ ) ₅ CH ₂ —
F (CF ₂ ) ₂ C (CH ₃ ) ₂ −
CF ₃ CHFCF ₂ C (CH ₃ ) ₂ −
F (CF ₂ ) ₆ CH ₂ —
F (CF ₂ ) ₅ (CH ₂ ) ₂ −
F (CF ₂ ) ₄ (CH ₂ ) ₃ −
(CF ₃ ) ₂ CF (CF ₂ ) ₂ (CH ₂ ) ₂ —
(CF ₃ ) ₂ CFCHFCF (CF ₃ ) CH _2-
CF ₃ CF ₂ CF (CF ₃ ) (CH ₂ ) ₃ −
H (CF ₂ ) ₆ CH ₂ —
_{Cl (CF 2) 6 CH 2} -
_{F (CF 2) 7 CH 2} -
F (CF ₂ ) ₆ (CH ₂ ) ₂ −
F (CF ₂ ) ₅ (CH ₂ ) ₃ −
F (CF ₂ ) ₄ (CH ₂ ) ₄ −
F (CF ₂ ) ₂ (CH ₂ ) ₆ −
F (CF ₂ ) ₃ OCF (CF ₃ ) (CH ₂ ) ₃ −
(CF ₃ ) ₃ C (CH ₂ ) ₄ −
_{H (CF 2) 7 CH 2} -
F (CF ₂ ) ₈ CH ₂ —
F (CF ₂ ) ₆ (CH ₂ ) ₃ −
(CF ₃ ) ₂ CF (CH ₂ ) ₆ −
(CF ₃ ) ₂ CF (CF ₂ ) ₄ (CH ₂ ) ₂ —
_{F (CF 2) 3 OCF (} CF 3) CF 2 OCF (CF 3) CH 2 -
H (CF ₂ ) ₈ CH ₂ —
F (CF ₂ ) ₄ (CH ₂ ) ₆ −
CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ −
F (CF ₂ ) ₈ (CH ₂ ) ₃ −
(CF ₃ ) ₂ CF (CF ₂ ) ₆ (CH ₂ ) ₂ —
H (CF ₂ ) ₁₀ CH ₂ —
F (CF ₂ ) ₆ (CH ₂ ) ₆ −
F (CF ₂ ) ₁₀ (CH ₂ ) ₂ −
H (CF ₂ ) ₁₂ CH ₂ —
F (CF ₂ ) ₈ (CH ₂ ) ₆ −
In formula (III), m is an integer of 1 to 3, preferably 2 to 3, and more preferably 1.

In formula (III), both X ^A are O or both are preferably S, and most preferably both are O.

In formula (III), B ² is the same or different and represents H, F, Cl, Br or I, and it is preferable that at least one of B ² is a halogen atom (F, Cl, Br or I). It is. Among these, it is preferable that ^two B ² in the formula (III) are both halogen atoms. Of the halogen atoms, a chlorine atom (Cl) and a fluorine atom (F) are preferable, and a fluorine atom (F) is more preferable. Particularly preferably, each of the ^two B ² in the formula (III) is a fluorine atom (F), and the form in which the above B ² is F (fluorine atom) is also the present invention. This is one of the preferred embodiments.

  From the viewpoint of the retention property of the neurosphere, in the above formula (II), Y consists of d-1 to d-10, e-1, f-1 to f-7, g-1 to g4 and the formula (III). It is preferably a structure selected from the group, more preferably a structure selected from the group consisting of d-3 and e-1, and still more preferably a structure represented by e-1.

In the above formula (II), Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ may be the same or different, and each independently represents a hydrogen atom or a fluorine atom. , A chlorine atom, a bromine atom or an iodine atom, and at least one of X 1 and Y does not contain a fluorine atom, at least one of Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ Is a fluorine atom. Although the number of fluorine atoms contained in Formula (II) should just be 1 or more, Preferably it is 4 or more. The upper limit of the number of fluorine atoms is not particularly limited and varies depending on the molecular structure, but is, for example, 30 or less per structural unit represented by the formula (II).

  From a viewpoint of retention of neurospheres, in a preferred embodiment, the structural unit represented by the above formula (II) is a structural unit represented by the following formula (II-2).

In the above formula (II-2), X is a group selected from the group consisting of an alkylene group optionally substituted with a fluorine atom, an aryleneoxy group, and an arylenethio group; Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ are the same as in the above formula (II); Z ⁷ , Z ⁸ , Z ⁹ , Z ¹⁰ , Z ^11, and Z ¹² are each independently a hydrogen atom, a fluorine atom, Any one of a chlorine atom, a bromine atom, an iodine atom, a methyl group and a trifluoromethyl group; X, Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , Z ⁶ , Z ⁷ , Z ⁸ , Z ⁹ , Z ¹⁰ , Z ¹¹ and Z ¹² each contain one or more fluorine atoms; Y ¹ is independently selected from an oxygen atom and a sulfur atom; q is an integer of 1 to 3, preferably 1 Or 2. Specific examples of X in the formula (II-2) are as described above for the formula (II).

  More specifically, the structural unit represented by the formula (II) is selected from structural units represented by the following formulas (II-3) (6FDA / TPEQ) and (II-4) (6FDA / ODA). More preferably.

  The fluorine-containing polyimide containing the structural unit represented by the above formula (II) can be obtained by a method of imidizing polyamic acid obtained by polymerization of acid dianhydride and diamine. Below, the synthesis | combination process of 6FDA / TPEQ copolymer is shown as one specific example.

  The polyamic acid synthesis reaction is preferably performed in an organic solvent. The organic solvent used in the polyamic acid synthesis reaction is not particularly limited as long as the reaction between the raw material acid dianhydride and the diamine can proceed efficiently and is inert to these raw materials. . For example, N-methylpyrrolidone (NMP), N, N-dimethylacetamide, N, N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, sulfolane, methyl isobutyl ketone, acetonitrile, benzonitrile, nitrobenzene, nitromethane, acetone, methyl ethyl ketone, isobutyl ketone And polar solvents such as methanol; nonpolar solvents such as toluene and xylene. Among these, it is preferable to use a polar solvent. These organic solvents may be used alone or as a mixture of two or more. The reaction mixture after the amidation reaction may be directly subjected to thermal imidation. The concentration of the polyamic acid in the polyamic acid solution is not particularly limited, but is preferably 5 from the polymerization reactivity and viscosity after polymerization of the resulting resin composition, subsequent film formation, and ease of handling in baking. % By weight or more, more preferably 10% by weight or more, preferably 50% by weight or less, more preferably 40% by weight or less.

  The polyamic acid is imidized by either thermal imidization or chemical imidization to obtain a resin composition containing a fluorine-containing polyimide.

  In a specific embodiment, the polyamic acid is imidized by heat treatment (thermal imidization) to obtain a resin composition containing a fluorine-containing polyimide. Polyimide obtained by thermal imidization has no possibility of remaining catalyst, and is more preferable for cell culture applications. The imidation ratio of the fluorine-containing polyimide resin may not be 100%. That is, the fluorine-containing polyimide resin partially includes a structural unit that is an amide structure in which a part of the cyclic imide structure of the structural unit represented by the above formula (I) or formula (II) is opened. May be.

  In the case of imidization by thermal imidization, for example, the polyamic acid is in the air, or more preferably in an inert gas atmosphere such as nitrogen, helium, or argon, or in vacuum, preferably at a temperature of 50 to 400 ° C. More preferably, the resin composition containing polyimide is obtained by performing an imidization reaction by baking under conditions of 100 to 380 ° C., preferably 0.1 to 10 hours, more preferably 0.2 to 5 hours. be able to.

  The polyamic acid subjected to the thermal imidization reaction is preferably in a form dissolved in a suitable solvent. Any solvent may be used as long as it dissolves polyamic acid, and the solvents described above for the polyamic acid synthesis reaction can also be used.

  In the case of imidization by chemical imidization, the polyamic acid can be directly imidized by using a dehydration cyclization reagent described later in a suitable solvent.

  The dehydrating cyclization reagent can be used without particular limitation as long as it has the action of chemically dehydrating and cyclizing polyamic acid to form a polyimide. As such a dehydrating cyclization reagent, the use of a tertiary amine compound alone or a combination of a tertiary amine compound and a carboxylic acid anhydride can promote imidization efficiently. Is preferable.

  Examples of the tertiary amine compound include trimethylamine, triethylamine, tripropylamine, tributylamine, pyridine, 1,4-diazabicyclo [2.2.2] octane (DABCO), 1,8-diazabicyclo [5.4. 0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene, N, N, N ′, N′-tetramethyldiaminomethane, N, N, N ′, N ′ -Tetramethylethylenediamine, N, N, N ', N'-tetramethyl-1,3-propanediamine, N, N, N', N'-tetramethyl-1,4-phenylenediamine, N, N, N ', N'-tetramethyl-1,6-hexanediamine, N, N, N', N'-tetraethylmethylenediamine, N, N, N ', N'-tetraethylethylenediamine And the like. Among these, pyridine, DABCO, N, N, N ′, N′-tetramethyldiaminomethane are particularly preferable, and DABCO is more preferable. Only one type of tertiary amine may be used, or two or more types may be used.

  Examples of the carboxylic acid anhydride include acetic anhydride, trifluoroacetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, succinic anhydride, maleic anhydride, and the like. Among these, acetic anhydride and trifluoroacetic anhydride are particularly preferable, and acetic anhydride is more preferable. Only one type of carboxylic anhydride may be used, or two or more types may be used.

As the solvent for dissolving the polyamic acid in the chemical imidization, a polar solvent having excellent solubility is suitable. For example, tetrahydrofuran, N, N-dimethylacetamide, N, N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide and the like can be mentioned, among which N, N-dimethylacetamide, N, N-dimethylformamide and N One or more selected from the group consisting of -methylpyrrolidone is preferable from the viewpoint of homogeneous reaction. When these solvents are used as the solvent for the amidation reaction, the polyamic acid can be used as it is for the chemical imidization without separation from the reaction mixture after the amidation reaction (fluorinated polyaryletherketone)
Examples of the fluorinated polyaryl ether ketone include fluorinated polyaryl ether ketones composed of structural units represented by the following formula (IV).

[In the formula (IV), n represents the degree of polymerization, m is an integer of 0 or 1, and R ⁴¹ represents the following formula (IV-2)

(In the formula (IV-2), p is an integer of 0 or 1, and R ⁴² has the following structure:

One of them. ). ]
Is a fluorinated polyaryletherketone represented by

  In the formula (IV), the degree of polymerization n is specifically 2 to 5000, preferably 5 to 500. Furthermore, in the present invention, the fluorinated polyaryletherketone may be composed of the same repeating unit or may be composed of different repeating units. In the latter case, the repeating unit is in the form of a block. Or any of a random form may be sufficient.

In the present invention, the production method of the fluorinated polyaryletherketone represented by the above formula (IV) will be described in detail. From this description, the terminal of the fluorinated polyaryletherketone represented by the formula (IV) is When the fluorine atom-containing benzene ring side is fluorine and the R ⁴¹ side is a hydrogen atom, that is, the fluorinated polyaryletherketone represented by the formula (IV) is represented by the following formula (IV-3):

[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 the fluorinated polyaryl ether ketone shown by these.

  In the above formula (IV), when m is 0, the following formula (IV-4):

[Wherein n represents the degree of polymerization. ]
The fluorinated polyaryletherketone represented by

  In the above formula (IV), when m is 1 and p is 0, the following formula (IV-5):

[Wherein n represents the degree of polymerization. ]
The fluorinated polyaryletherketone represented by

  Furthermore, in the above formula (IV), when m is 1 and p is 1, the following formula (IV-6):

[Wherein n represents the degree of polymerization, and R ⁴² is as defined above. ]
The fluorinated polyaryletherketone represented by In the above formula (IV-6), n is preferably 2 to 2000, more preferably 5 to 200.

  That is, in a preferred embodiment, the fluorinated polyaryletherketone represented by the formula (IV) is represented by the following formula (IV-7):

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

  In a particularly preferred embodiment, the fluorinated polyaryletherketone of formula (IV) has the following formula (IV-8):

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

  Among these, the fluorinated polyaryl ether ketones represented by the formulas (IV-2) and (IV-4) are represented by the following formula (IV-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

  In the above reaction, the reaction temperature is 30 to 250 ° C, preferably 50 to 200 ° C.

  In addition, the fluorinated polyaryletherketone represented by the above (IV-6) is represented by the following formula (IV-10) 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 (IV-11):

[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 performed under normal pressure or under reduced pressure, but it is preferable to perform 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 (IV-2) and (IV-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 (IV-5), preferably 1-20 mol, preferably 1-20 mol, relative to 1 mol of the pentafluorobenzoyldiphenyl 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. As a result, a fluorinated polyaryletherketone containing a repeating unit represented by the above formula (IV-8) 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 adding the reaction solution to a solvent having low polymer solubility to precipitate the polymer as a solid and separating the precipitate by filtration.

(Fluorinated polyester polyether)
As a fluorinated polyester polyether, the fluorinated polyester polyether which consists of a structural unit shown by the following formula | equation (V) can be illustrated.

[In Formula (V), 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. ]
Is a fluorinated polyester polyether essentially comprising a repeating unit represented by

  The fluorinated polyester polyether used in the present invention may contain other repeating units as long as the repeating unit represented by the above formula (V) is essential, but in the above formula (V), The repeating unit represented is preferably the main component of the repeating unit constituting the fluorinated polyester polyether. In the fluorinated polyester polyether, the structure of the repeating unit represented by the above formula (V) may be the same or different. Any form such as a random shape may be used.

In the repeating unit represented by the above formula (V), the (—O—R ⁵² —O—) moiety may be any of the ortho, meta, and para positions with respect to the carbon bonded to the ester of the benzene ring. It may be bonded to carbon, but is preferably bonded to the ortho or para position. In the fluorinated polyester polyether used in the present invention, the benzene ring having a fluorine atom has a form in which some or all of the four hydrogen atoms are substituted with fluorine atoms, and the hydrogen atoms of the benzene ring May be substituted with other substituents such as a halogen atom other than a fluorine atom or a substituent having an alkyl chain. Therefore, the sum 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 represent a substituent, and represent one benzene ring Has 0-4 Y ¹ , Y ² , Y ³ and Y ⁴ , 0-3 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 fluorinated polyester polyether used in the present invention, the benzene ring of the groups represented by the above formulas (8-1) to (8-19) has one or more substituents, and the substituents have carbon atoms. It is 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.

In the fluorinated polyester polyether used in the present invention, in the above formula (V), the structure of R ⁵¹ is any one of the above (9-6) or (9-18), and the benzene ring is a substituent. It is preferable that it does not have. That is, the fluorinated polyester polyether has the following formula (V-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 (V-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.

  Although the manufacturing method in particular of the fluorinated polyester polyether represented by the said Formula (V) 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.

  That is, the fluorinated polyester polyether represented by the above formula (V) is represented by the following formula (V-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 (V-5):

In the formula, 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 (V-4) has high reactivity, when a polymer is produced using 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, it is preferably bonded to the ortho or para position. 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 fluorine-containing 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. Those having an action of changing to a highly reactive anion are suitable, for example, calcium carbonate, calcium hydroxide, potassium fluoride, tributylamine, pyridine, potassium carbonate, lithium carbonate, potassium hydroxide, triethylamine and the like 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 if necessary, whereby a fluorinated polyester polyether having a repeating unit represented by the above formula (V) is obtained. Will be obtained. Alternatively, the fluorinated polyester polyether can be precipitated as a solid by adding the reaction solution in a solvent having a low polymer solubility, and the precipitate can be separated by filtration.
(Fluorinated polyether)
Examples of the fluorinated polyether include fluorinated polyethers composed of structural units represented by the following formula (VI).

[In Formula (VI), R ³¹ represents 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 atom which may have a substituent Represents an arylthio group of 6 to 20; R ⁶¹ represents a divalent organic group; and n represents a degree of polymerization. ]
It is a fluorinated polyether shown by.

In the formula (VI), R ³¹ is an alkyl group having 1 to 12 carbon atoms which may have a substituent, for example, 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 12 carbon atoms 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-butyl Amino, 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; optionally substituted aryl groups having 6 to 20 carbon atoms such as phenyl, 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; an 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 ester, furfuryl ester and phenyl ester; the same shall apply hereinafter), naphthoxy, o-, m- or p-methylphenoxy, o-, m- or p-phenylphenoxy, phenylethynylphenoxy, and Cresotic acid And groups derived from esters thereof, 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 having substituents An arylthio group having 6 to 20 carbon atoms which may be represented, for example, phenylthio, phenylmethanethio, o-, m- or p-tolylthio, and a group 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 .

In the formula (VI), a substituent that can be used when R ³¹ represents a substituted alkyl group, alkoxy group, alkylamino group, alkylthio group, aryl group, aryloxy group, arylamino group or arylthio group. Can be appropriately selected according to the desired properties of the target product, and is not particularly limited. For example, an 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 and dodecyl; halogen atoms such as fluorine, chlorine, bromine and iodine; cyano group, nitro group and carboxy ester Group and the like. Of these, methyl and carboxyester groups are preferred.

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

  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 (VI), n represents a polymerization degree, and is specifically 5-1000, Preferably it is 10-500. In addition, the fluorinated polyether used in the present invention may be composed of the same repeating unit of the structural unit of the above formula (VI) or may be composed of different repeating units. The repeating unit may be either a block shape or a random shape.

Further, the production method of the fluorinated polyether will be described in detail below. From this description, the terminal of the fluorinated polyether represented by the formula (VI) is fluorine on the benzene ring side containing the fluorine atom, and the oxygen atom. When the (R ⁶¹ ) side is a hydrogen atom, that is, the fluorinated polyether represented by the formula (VI) is represented by the following formula (VI-4):

It is thought that it is a polymer shown by.

  The fluorinated polyether has the following formula (VI-2):

A tetrafluorobenzonitrile derivative represented by the following formula (VI-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 (VI-2) and R ^{61 in} the above formula (VI-3) is the same as the definition of R ³¹ and R ⁶¹ in the above formula (VI).

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

In the above reaction, the compound represented by the formula: R ³¹ H and PFBN may be used as a single compound, respectively, or a mixture of two or more compounds represented by the formula: R ³¹ H and / or PFBN 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 is equal to or substantially equal to the sum of the 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 R1H and PFBN proceeds efficiently. For example, the reaction is preferably performed while the reaction system is in a stirred state. While maintaining, it is usually carried out 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 adding distilled water to 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 (VI-2) synthesized in this way is further subjected to polymerization in the presence of a dihydroxy compound of the formula (VI-3) and a basic catalyst as described above. To produce the desired fluorinated polyether of formula (VI). At this time, the tetrafluorobenzonitrile derivative of the formula (VI-2) may be used after being subjected to a purification step such as extraction, recrystallization, chromatography and distillation as described above or without performing the purification step. 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 (VI-3) used in the above reaction is selected according to the structure of the fluorinated polyether of formula (VI) which is the target product. As the dihydroxy compound of the formula (VI-3) preferably used, 2,2-bis (4-bidoxyphenyl) -1,1,1,3,3,3-hexahexane is used 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'-dihydroxybifu Nil (hereinafter referred to as "BP"), and the like.

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

  The 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, usable organic solvents include, for example, 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 (VI-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 the formula (VI-2) and the dihydroxy compound of the formula (VI-3) must be carried out in the presence of a basic catalyst. The basic catalyst is preferably one having an action of converting the dihydroxy compound of the formula (VI-3) into a more reactive anion so as to promote the polycondensation reaction with the dihydroxy compound of the formula (VI-3). Examples include potassium carbonate, calcium carbonate, potassium hydroxide, calcium hydroxide, or potassium fluoride. The amount of the basic catalyst used is not particularly limited as long as the reaction between the tetrafluorobenzonitrile derivative of the formula (VI-2) and the dihydroxy compound of the formula (VI-3) can proceed satisfactorily. However, it is usually 0.1 to 5 mol, preferably 0.5 to 2 mol, per 1 mol of the tetrafluorobenzonitrile derivative of the formula (VI-2).

  Furthermore, the reaction conditions in the polymerization reaction are not particularly limited as long as the reaction between the tetrafluorobenzonitrile derivative of the formula (VI-2) and the dihydroxy compound of the formula (VI-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 performed under normal pressure or under reduced pressure, but it is preferable to perform 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.

(Properties of fluorine-containing polymer)
The fluorine content of the fluoropolymer used in the present invention is, for example, 1 to 60% by mass, preferably 5 to 60% by mass, more preferably 10 to 60% by mass, still more preferably 15 to 30% by mass, and particularly preferably. It is 17-19 mass% or less. In addition, said fluorine content can be measured by the method as described in an Example.

  The cell culture surface of the substrate preferably has a static water contact angle of 75 ° or more, more preferably more than 80 °, still more preferably more than 83 °, and the upper limit of the static water contact angle is, for example, 150 It is less than °, preferably less than 120 °. In addition, said static water contact angle is a value measured by the method as described in an Example.

  As the fluorine-containing polymer used in the present invention, those having a tensile elastic modulus exceeding 2 GPa are preferable. By using a fluorine-containing polymer having a tensile elastic modulus of 1 GPa or more, the retention of neurospheres is further improved. The tensile modulus of the fluorinated polymer is more preferably more than 2 GPa, still more preferably 2.3 GPa or more. The upper limit of the tensile elastic modulus is not particularly limited, but actually, for example, it is 6 GPa or less, preferably 4 GPa or less, more preferably less than 3.4 GPa, and further preferably 3 GPa or less. In a preferred embodiment, a fluoropolymer having a tensile modulus of more than 2 GPa and less than 3.4 GPa is used. The tensile elastic modulus of the fluoropolymer can be controlled by any means. For example, when there are many ether bonds in the polymer main chain, a flexible structure can be obtained. Therefore, the tensile elastic modulus can be increased by reducing the number of groups that give flexibility to molecules such as ether bonds per molecule of the polymer by a method such as selecting a material or lowering the degree of polymerization. . Alternatively, for example, in the case of fluorine-containing polyimide, the tensile elastic modulus is controlled by appropriately setting the conditions for the imidization reaction (for example, the tensile elastic modulus can be increased by increasing the firing temperature). Can do. In addition, said tensile elasticity modulus is a value measured by the method as described in an Example.

  The weight average molecular weight of the fluorine-containing polymer is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, more preferably 10,000 to 500,000, and still more preferably. Is 100,000 or more and less than 300,000. In addition, in this specification, the weight average molecular weight of a polymer is the value measured by the method as described in an Example.

  The culture vessel used for culture is not particularly limited as long as the cell adhesion surface is composed of a fluoropolymer substrate. The cell culture vessel may be configured by combining a fluoropolymer base material and another member, or may be configured by integrating the fluoropolymer base material and another member. Further, it may be composed of only a fluoropolymer substrate. When the fluorine-containing polymer substrate is a flexible substrate such as a film, it may be formed in combination with an appropriate support member having rigidity. Examples of the material constituting the supporting member include inorganic glass such as soda glass, alkali-free glass, and borosilicate glass; carbon; metal such as silicon; polyolefin resin such as polyethylene, polypropylene, and cyclic olefin; polyethylene terephthalate (PET) Polyester resins such as polymethyl methacrylate; epoxy resins; polyvinyl chloride, polyvinylidene chloride, polystyrene resins, polyvinyl acetate, ABS (acrylonitrile-butadiene-styrene) resins, polycarbonate resins, vinyl ethers, polyacetals, Polyphenylene ether (PPE), polyaryl ether, polyphenylene sulfide (PPS), polyether ether ketone (PEEK), polyaryl ether ketone, phenol resin Polyether nitrile (PEN), silica and the like. The cell culture container may have any shape as a whole as long as the cell adhesion surface includes a fluoropolymer substrate. For example, it can be in the form of various plates such as plates for culture such as single or multiwell plates, petri dishes, dishes, flasks, or bags. The cell culture container 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.

(Neural stem cells)
In this specification, a neural stem cell is a cell that has the ability to differentiate into nerve cells and glial cells (for example, astrocytes, oligodendrocytes, etc.) and proliferate (self-replicating ability). The biological species from which the neural stem cells are derived is not particularly limited, and various cells derived from humans and non-human animals can be used. Examples of the biological species from which the cells are derived include, for example, primates such as humans, rhesus monkeys, green monkeys, cynomolgus monkeys, chimpanzees, tamarins and marmosets, rodents such as mice, rats, hamsters and guinea pigs, dogs, cats, rabbits, pigs, Examples include cows, goats, sheep, horses, chickens, quails, minks, pineapples, Xenopus laevis, and zebrafish. Neural stem cells may also be collected from living organisms, or totipotent cells, or induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells), etc. Pluripotent cells may be induced to differentiate into neural stem cells by the method described in International Publication No. 2011/108766. Differentiation induction of totipotent cells or pluripotent cells into neural stem cells may be performed on the fluoropolymer substrate according to the present invention.

  When neural stem cells are collected from a living tissue and used, the tissue from which the stem cells are derived is not particularly limited and may be either a central nervous system tissue or a peripheral nervous system tissue. For example, neural stem cells derived from the brain are preferably used. Can be.

  In this specification, a neurosphere is a cell aggregate containing neural stem cells. The size of the neurosphere formed (the size of the neurosphere before differentiation induction) is, for example, 10 to 1000 μm in diameter, preferably 50 to 500 μm, and more preferably 100 to 200 μm. In addition, said magnitude | size of neurosphere is an average value about ten or more neurospheres observed with the optical microscope.

  When neurospheres are formed by conventional suspension culture, excessively large neurospheres may be formed due to the association of the neurospheres floating in the medium. In this case, there exists a possibility that the cell which exists in the neurosphere center part may be necrotized. For this reason, in order to prevent the formation of excessively large neurospheres, conventionally, measures such as suspension culture in a minute well having a diameter of about 400 μm have been taken. On the other hand, according to the present invention, since the neurospheres are held on the base material, the association of the neurospheres due to floating can be effectively suppressed. Thereby, it is possible to effectively prevent the neurosphere from becoming excessively large without requiring a means having poor operability such as culturing in a minute well.

  In the present invention, cells containing at least neural stem cells may be cultured on a fluoropolymer substrate. That is, the presence of cells other than neural stem cells (for example, neural progenitor cells, glioblasts, etc.) in the neurosphere is allowed to the extent that the objective effect of the present invention is not inhibited. In the present invention, neural stem cells are cells other than neural stem cells (for example, pluripotent cells such as iPS cells and ES cells; mesenchymal stem cells, hematopoietic stem cells, hepatic stem cells, pancreatic stem cells, etc.) Cells: Adipocytes, hepatocytes, kidney cells, pancreas cells, mammary cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, cardiomyocytes, dendritic cells, chondrocytes, bone, including various progenitor cells and stem cells Neurosphere formation in the presence of blast cells, osteoclasts, bone cells, fibroblasts, various blood cells, retinal cells, corneal cells, gonad cells, various line cells, various cancer cells, etc.) Also good. The cells other than the neural stem cells may be derived from the same tissue as the neural stem cells or may be derived from other tissues.

  As a basal medium used for culturing neural stem cells, a conventionally known medium usually used for culturing neural stem cells can be adopted, and is not particularly limited. For example, as a basal medium, Eagle minimum essential medium (E-MEM), Dulbecco's modified Eagle medium (D-MEM), α-MEM, Glasgow MEM (G-MEM), IMDM, RPMI 1640, HAM'sF-10 medium ( F-10), HAM'sF-12 medium (F-12), MCDB medium, Williams medium E, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium and the like. As the basal medium, a mixed medium in which the above medium is mixed at an arbitrary ratio, such as D-MEM / F-12, may be used. Furthermore, as necessary, growth factors, antibiotics, lipids, hormones (eg, insulin, dexamethasone, hydrocortisone, β-estradiol, progesterone, etc.), iron sources (eg, transferrin, etc.), organic acids (eg, pyruvate, etc.) ), Amino acids (eg, L-glutamic acid, non-essential amino acids, etc.), sugars (eg, glucose, heparin, etc.), vitamins (eg, ascorbic acid, biotin, etc.), salts (eg, sodium selenite, etc.), reducing agents (For example, 2-mercaptoethanol), ethanolamine, serum protein (for example, albumin), buffer (for example, HEPES), various supplements for cell culture (for example, N2-supplement, ITS-supplement, GlutaMAX-I) Supplements), BMP inhibitors ( In example it may be used medium supplemented with additives such as Noggin, etc.).

  The growth factor added to the medium is not particularly limited as long as it can be used at a concentration that promotes proliferation of neural stem cells and does not induce differentiation. From the viewpoint of cell proliferation, neural stem cells are cultured from the group consisting of EGF (epidermal growth factor), bFGF (basic fibroblast growth factor), HGF (hepatocyte growth factor), and LIF (leukemia inhibitory factor). It is preferably performed in the presence of one or more selected growth factors. The addition amount of these growth factors may be appropriately adjusted depending on the cell density or the like. For example, the concentration of each growth factor in the medium is 1 to 100 ng / ml, preferably 10 to 40 ng / ml.

  In the present invention, from the viewpoint that differentiation induction is effectively suppressed, neural stem cells are preferably cultured in a serum-free medium. In this specification, the serum-free medium means a basal medium that does not contain unconditioned or unpurified serum, and a medium that contains purified blood-derived components or animal tissue-derived components (for example, growth factors) Corresponds to serum-free medium. In particular, from the viewpoint of proliferation, the medium used for the culture of neural stem cells is preferably a serum-free medium containing N2-supplement (Bottensin, J. et al .: Methods Enzymol., 58, 94 (1979)). .

Hereinafter, as one specific example, the composition of a suitable medium used for culturing neural stem cells is shown:
D-MEM / F-12 mixed medium 10-40 ng / ml EGF
10-40 ng / ml bFGF
1-50 μg / ml heparin 0.5-4% (v / v) N-2 supplement.

The cell density at the start of culture of neural stem cells on the fluoropolymer substrate (density of whole cells including neural stem cells and other cells) is, for example, 1 × 10 ² to 1 × 10 ⁶ cells / cm ² , It is preferably 1 × 10 ^{3 to} 5 × 10 ⁵ cells / cm ² .

The culture conditions are not particularly limited, and the conditions are those usually used in this technical field. For example, the culture conditions are 1 to 10% (v / v), preferably about 5% (v / v) in a CO ₂ atmosphere at about 25 to 40 ° C., preferably 32 to 38 ° C. This is performed until neurospheres are formed (for example, about 3 to 15 days). It is preferable to change the medium at a pace of 2 to 5 days. The medium exchange may be a full exchange or a partial exchange (for example, a half exchange).

  Presence of undifferentiated neural stem cells in the formed cell aggregate can be confirmed by expression of a neural stem cell gene or protein molecular marker. As such a neural stem cell molecular marker, a conventionally known one can be used. For example, a plurality of molecular markers such as an intermediate filament such as nestin may be measured in combination as necessary. The molecular marker may be measured by a conventionally known means such as PCR (for example, quantitative PCR) or Northern blotting if it is a gene, or ELISA or immunostaining if it is a protein.

<Method of forming neurospheroid>
In one aspect of the present invention, the neurospheroid includes: forming neurospheres by the neural stem cell culture method described above; and inducing differentiation of the neural stem cells in a state where the neurospheres are retained on the base material. A forming method is provided. By forming a neurospheroid through the formation of neurospheres, a neurospheroid in which a three-dimensional network is formed in an arbitrary size and shape can be obtained. In particular, when an induction treatment using an inducer is performed on neurospheres retained on a fluoropolymer, cells respond to the inducer with high sensitivity and differentiation can be strongly induced.

  The neuropheroid is an aggregate formed by a plurality of cells including nerve cells, glial cells (for example, astrocytes, oligodendrocytes, etc.) and / or their precursor cells. Neurospheroids are undifferentiated cells such as neural stem cells, cells of tissues other than the nervous system (for example, pluripotent cells such as iPS cells and ES cells; mesenchymal stem cells, hematopoietic stem cells, hepatic stem cells, pancreatic stem cells, etc. Multipotent cells: including various progenitor cells and stem cells, adipocytes, hepatocytes, kidney cells, pancreatic cells, mammary cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, cardiomyocytes, dendrites Cells, chondrocytes, osteoblasts, osteoclasts, bone cells, fibroblasts, various blood cells, retinal cells, cornea-derived cells, gonad-derived cells, various line cells; various cancer cells, etc.) You may go out. The neuropheroid may contain any of nerve cells and glial cells, and may contain both. In one embodiment, neurospheroids comprising neural cells are provided, and in another embodiment, neurospheroids comprising glial cells are provided. The shape and size of the neuropheroid are not particularly limited, and can be appropriately set according to the use and purpose of use. For example, the diameter (excluding neurites) is 10 to 1000 μm, preferably 50 to 500 μm, more preferably 100. ~ 200 μm. In addition, the magnitude | size of said neuro spheroid is an average value about ten or more neuro spheroids observed with the optical microscope.

  Differentiation induction of neural stem cells is usually performed by culturing in a medium containing an inducer (differentiation inducer). By appropriately selecting the inducer, it is possible to obtain desired cells with high selectivity. The basal medium used for inducing differentiation of neural stem cells is not particularly limited. For example, Eagle small essential medium (E-MEM), Dulbecco's modified Eagle medium (D-MEM), α-MEM, Glasgow MEM (G- MEM), IMDM, RPMI 1640, HAM'sF-10 medium (F-10), HAM'sF-12 medium (F-12), MCDB medium, Williams medium E, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium Etc. As the basal medium, a mixed medium in which the above medium is mixed at an arbitrary ratio, such as D-MEM / F-12, may be used. Further, as necessary, growth factors (eg, EGF, FGF (eg, bFGF), IGF, HGF, TGF-β, etc.), antibiotics, lipids, hormones (eg, insulin, dexamethasone, hydrocortisone, β-estradiol, Progesterone etc.), iron sources (eg transferrin etc.), organic acids (eg pyruvic acid etc.), amino acids (eg L-glutamic acid, non-essential amino acids etc.), sugars (eg glucose, heparin etc.), vitamins (eg , Ascorbic acid, biotin, etc.), salts (eg, sodium selenite, etc.), reducing agents (eg, 2-mercaptoethanol, etc.), ethanolamine, serum proteins (eg, albumin, etc.), buffers (eg, HEPES, etc.) ), Various cell culture supplements (eg, N2-supplements, Media supplemented with additives such as ITS-supplements, GlutaMAX-I supplements, etc.) may be used.

  As the inducer (differentiation inducer), conventionally known ones can be appropriately selected and used. More specifically, neural stem cell differentiation is induced by, for example, serum (eg, animal serum such as fetal bovine serum, calf serum, horse serum, pig serum, goat serum, sheep serum, rabbit serum, dog serum). , B27-supplement, SM-Neuronal supplement, retinoic acid and its derivatives (eg, retinoic acid, retinol, retinal, 3-dehydroretinoic acid, 3-dehydroretinol, 3-dehydroretinal, AM580 (4-[[((5 6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carbonyl] amino] -benzoic acid), TTNPB (4-[(1E) -2- (5,6,7, 8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-propen-1-yl] -benzoic acid), Retinol mycinate, etc.), NGF (nerve growth factor), LIF (leukemia inhibitory factor), GDNF (glial cell-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), BDNF (brain-derived neurotrophic factor), NT -3 (neurotrophin-3), BMP2 (bone morphogenetic protein-2), BMP4 (bone morphogenetic protein-4), and a medium containing one or more selected from the group consisting of SHH (sonic hedgehog) By culturing. One inducer (differentiation inducer) may be used alone, or two or more inducers may be used in combination, and the composition may be appropriately changed according to the degree of differentiation. As a method for inducing differentiation into nerve cells, differentiation induction of neural stem cells is carried out in a medium containing B27-supplement (Brewer, GJ et al .: J. Neurosci. Res., 35, 567 (1993)). More preferably, it is more preferably carried out by culturing in a Neurobasal medium containing B27-supplement. Although the detailed mechanism is unknown, when a fluorine-containing polymer is used as a base material, the responsiveness of the differentiation signal by the inducing agent is improved as compared with the suspension culture. In particular, by using the B27 supplement, the differentiation signal In particular, the responsiveness is significantly improved. The addition amount of B27-supplement is, for example, 1 to 4% (v / v) in the medium.

The culture conditions are not particularly limited, and the conditions are those usually used in this technical field. For example, the culture conditions are 1 to 10% (v / v), preferably about 5% (v / v) in a CO ₂ atmosphere at about 25 to 40 ° C., preferably 32 to 38 ° C. Until the neurosphere is formed (for example, about 1 to 20 days). It is preferable to change the medium at a pace of 1 to 5 days. The medium exchange may be a full exchange or a partial exchange (for example, a half exchange).

  Neural stem cell differentiation can also be confirmed by the expression of molecular markers such as genes, proteins, and sugars. As such differentiation markers, conventionally known markers can be appropriately selected and used. For example, PSA-NCAM (polysialic acid neural adhesion molecule), β tubulin-3, NeuN, GFAP (glial cell fibrillary acidity) The molecular markers such as protein), PLP (proteolipid protein), MBP (myelin basic protein) and the like may be appropriately combined as necessary. For the measurement of molecular markers, conventionally known means such as PCR (for example, quantitative PCR) and Northern blotting can be used for genes, and ELISA methods and immunostaining methods can be used for proteins.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.

<Example 1>
(Measurement method of fluorine content)
The fluorine content in the fluorine-containing polymer film was quantified with an elemental analyzer (manufactured by J Science: Microcoder JM-10).

(Measurement of weight average molecular weight)
Device: HCL-8220GPC manufactured by Tosoh Corporation
Column: TSKgel Super AWM-H
Eluent (LiBr · H ₂ O, NMP with phosphoric acid): 0.01 mol / L
Measurement method: A 0.5 wt% solution was prepared with an eluent, and the molecular weight was calculated based on a calibration curve prepared with polystyrene.

(Measurement of static 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 dropping 2 μl of water on the film was measured (measurement temperature: 25 ° C.).

(Dynamic viscoelasticity measurement method)
Apparatus: Dynamic viscoelasticity (manufactured by TA Instruments Inc .: RSA G2)
Measurement method: A film having a thickness of 40 μm was cut into a strip of 5 mm × 40 mm, the elongation and stress at 25 ° C. were measured, and the tensile modulus was calculated.

(Manufacture of fluoropolymer substrate)
In a 100 ml three-necked flask, 2.976 g (10.2 mmol) of 1,4-bis (aminophenoxy) benzene (TPEQ), 4.524 g of 4,4′-hexafluoroisopropylidenediphthalic anhydride (6FDA) ( 10.2 mmol) and 42.5 g of N, N-dimethylacetamide were charged. By stirring at room temperature for 5 days under a nitrogen atmosphere, a fluorine-containing polyamic acid resin composition (solid content concentration: 15.0% by mass) was obtained. The weight average molecular weight of the polyamic acid was 180,000. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorinated polyimide after firing are substantially the same.

  A coating film was formed on the glass substrate using a die coater so that the thickness of the fluorine-containing polyimide film after baking the fluorine-containing polyamic acid resin composition obtained above was 40 μm. Next, the coating film was baked at 300 ° C. for 1 hour in a nitrogen atmosphere. Thereafter, the fired product was peeled from the glass substrate to obtain a fluorine-containing polyimide film 1.

  The obtained fluorine-containing polyimide film 1 had a fluorine content of 17% by mass, a static water contact angle of 87 °, and a tensile elastic modulus of 2.8 GPa.

(Neural stem cell culture)
Using the fluorine-containing polyimide film 1 obtained above as a base material, a 24-well plate arranged on the cell culture surface was prepared and subjected to neural stem cell culture by the following method. The fluorine-containing polyimide film was sterilized and used for cell culture.

A 14-day-old fetus was removed from the Wistar rat, and the brain was collected from the removed fetus. The collected fetal brain was treated with 2 ml of a cell separation / dispersion solution (Accumax, manufactured by Innovative Cell Technologies) for 10 to 15 minutes at room temperature to obtain a cell dispersion. Next, 4 ml of D-MEM / F-12 (1: 1 (v: v)) medium (Sigma-Aldrich) was added to the obtained cell dispersion, and the cells were filtered with a filter having a pore size of 30 μm. The solution was centrifuged at 900 rpm for 5 minutes. After centrifugation, the supernatant was removed and D- containing maintenance medium (1% (v / v) N2-supplement (Life technologies), 20 ng / ml human bFGF, 20 ng / ml human EGF, and 10 μg / ml heparin. The precipitate was resuspended in MEM / F-12 (1: 1 (v: v)) medium (Sigma-Aldrich)) to a concentration of 2.5 × 10 ⁵ cells / ml. The cell suspension prepared above was seeded (5 × 10 ⁴ cells / cm ² ) in a 24-well plate in which the fluoropolymer substrate was placed on the cell culture surface (culture day 0). Thereafter, the cells were cultured in a 5% (v / v) CO ₂ incubator at 37 ° C., and the medium was changed by changing the half amount on the third day of culture, and cultured until the eighth day. It was confirmed with an optical microscope that the neurospheres retained on the base material appeared with the progress of the culture. The observation image by an optical microscope is shown in FIG. 1 (FIG. 1 (a) culture 1st day, (b) culture 3 days, (c) culture 5 days, (d) culture 8 days). The size of the neurosphere on the 8th day of culture was 171 ± 66 μm in diameter (average value of 41 neurospheres ± standard deviation μm).

(Confirmation of undifferentiation)
Cells were collected from the culture plate on which neural stem cells were cultured using a cell separation / dispersion solution (Accummax, manufactured by Innovative Cell Technologies). The recovered cells were washed with Neurobasal medium containing B-27 supplement. The collected cells were suspended in 1 ml of Neurobasal medium containing B-27 supplement, and the number of cells was counted. RNA was recovered from the obtained cells using illustra RNAspin Mini RNA Isolation Kit (GE Healthcare).

  Using the recovered RNA as a sample, gene expression levels of nestin (undifferentiation marker) and β-tubulin-3 (differentiation marker) were measured by quantitative PCR. In addition, TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Applied Biosystems) were used as kits for quantitative PCR, and the measuring instrument name was 7300/7500 Realtime PCR Systems (April). The gene expression levels of nestin and β-tubulin-3 are calibrated as relative amounts with respect to the expression levels of GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and the relative expression levels of each target gene in Comparative Example 1 are Calculated as 1. The results are shown in FIG.

(Neurospheroid formation)
On day 8 of culture, after confirming the formation of neurospheres, the medium was replaced with a differentiation medium (Neurobasal medium (Life technologies) containing 2% (v / v) B27-supplement (Life technologies)). The culture was continued while the medium was changed by half each day, and it was confirmed with a light microscope that neuropheroids were formed.

  Fig. 2 (Fig. 2 (a) Day 1 of culture medium replacement on differentiation medium, day 0 of culture on differentiation medium, and (b) Day 3 of culture in differentiation medium) (C) Day 5 of culture in differentiation medium, (d) Day 9 of culture in differentiation medium). As shown in FIG. 2, when a fluoropolymer substrate was used, a three-dimensional network structure in which neurospheroids were linked was confirmed after the fifth day of culture in a differentiation medium.

  On the 11th day (19 days after seeding of neural stem cells) after replacement with the differentiation medium, cells were collected by the same method as described above using a cell separation / dispersion solution (Accummax, manufactured by Innovative Cell Technologies), and RNA was recovered. It was collected.

  Using the recovered RNA as a sample, the gene expression levels of nestin and β-tubulin-3 were measured by the quantitative PCR method in the same manner as described above. The measurement results of the marker molecule expression level are shown in FIG.

<Comparative Example 1>
Neural stem cells were cultured in the same manner as in Example 1 except that a floating culture plate in which the cell adhesion to the substrate was reduced by surface treatment of the cell culture substrate surface was used. The observation images of neural stem cells cultured using the suspension culture plate with an optical microscope are shown in FIG. 3 (FIG. 3 (a) culture day 1, (b) culture day 3, (c) culture day 5, ( d) 8th day of culture). In the suspension culture system culture plate, the neurospheres were not retained on the base material but suspended in the medium. In addition, the size of the neurosphere was enlarging compared to the case of culturing on a fluoropolymer substrate.

  Neurospheres were collected from the suspension culture plate in which neural stem cells were cultured using a cell separation / dispersion solution (Accumax, manufactured by Innovative Cell Technologies), and RNA was collected in the same manner as in Example 1.

  On the 8th day of culture, culture in differentiation medium was started in the same manner as in Example 1 except that a floating culture plate was used instead of the plate using the fluoropolymer substrate.

  FIG. 4 shows an image of neural stem cells cultured in a differentiation medium using a suspension culture plate, as observed with an optical microscope (FIG. 4 (a). (B) culture day 3 in differentiation medium, (c) culture day 5 in differentiation medium, (d) culture day 9 in differentiation medium). As shown in FIG. 4, when cultured in suspension culture, it was not possible to confirm a three-dimensional network structure in which neurospheroids were linked.

  On the 11th day (19 days after seeding of neural stem cells) after replacement with the differentiation medium, cells were collected by the same method as described above using a cell separation / dispersion solution (Accummax, manufactured by Innovative Cell Technologies), and RNA was recovered. It was collected.

  Using the recovered RNA as a sample, the gene expression levels of nestin and β-tubulin-3 were measured by the quantitative PCR method in the same manner as described above. The measurement results of the marker molecule expression level are shown in FIG.

  As shown in FIG. 5, in Comparative Example 1, the increase in the expression level of the differentiation marker was not large even after the medium was changed to the differentiation medium.

  As shown in FIG. 5, in Example 1, the high expression level of the undifferentiation marker before differentiation induction and the low expression level of the differentiation marker were confirmed. This indicates that there are many neural stem cells in the neurosphere held on the base material, and differentiation induction is effectively suppressed. Furthermore, in Example 1, the expression level of the differentiation marker was significantly increased after differentiation induction. From this result, it is shown that the cells respond to the differentiation induction signal with high sensitivity by performing differentiation induction treatment on the neurospheres retained on the fluoropolymer substrate.

<Examples 2 and 3>
(Manufacture of fluoropolymer substrate)
In a 100 ml three-necked flask, 2.330 g (11.64 mmol) of 4,4′-diaminodiphenyl ether (ODA) and 42.5 g of N, N-dimethylacetamide were charged and dissolved. Thereto was added 4.170 g (11.64 mmol) of 4,4′-hexafluoroisopropylidenediphthalic anhydride (6FDA), and the mixture was stirred at room temperature for 5 days in a nitrogen atmosphere, whereby a fluorine-containing polyamic acid resin was obtained. A composition (solid content concentration 15.0 mass%) was obtained. The weight average molecular weight of the polyamic acid was 120,000. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorinated polyimide after firing are substantially the same.

  Using the fluorine-containing polyamic acid resin composition obtained above, a fluorine-containing polyimide film 2 was obtained in the same manner as in Example 1.

  The obtained fluorine-containing polyimide film 2 had a fluorine content of 19% by mass, a static water contact angle of 86 °, and a tensile elastic modulus of 2.9 GPa.

  In a 100 ml three-necked flask, 3.141 g (9.81 mmol) of 2,2′-bis (trifluoromethyl) benzidine (TFMB) and 42.5 g of N, N-dimethylacetamide were charged and dissolved. Thereto, 4.359 g (9.81 mmol) of 4,4′-hexafluoroisopropylidenediphthalic anhydride (6FDA) was added, and the mixture was stirred at room temperature for 5 days in a nitrogen atmosphere. A composition (solid content concentration of 15% by mass) was obtained. The weight average molecular weight of the polyamic acid was 300,000. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorinated polyimide after firing are substantially the same.

  Using the fluorine-containing polyamic acid resin composition obtained above, a fluorine-containing polyimide film 3 was obtained in the same manner as in Example 1.

  The resulting fluorine-containing polyimide film 3 had a fluorine content of 31% by mass, a static water contact angle of 88 °, and a tensile elastic modulus of 3.4 GPa.

(Neural stem cell culture)
24-well plates were prepared by placing the fluorine-containing polyimide film 2 and the fluorine-containing polyimide film 3 obtained above on the cell culture surface, respectively, and neural stem cells were cultured by the same method as in Example 1. Neurospheres in the culture process were observed with an optical microscope and evaluated according to the following criteria. The evaluation results are shown in Table 1 below.
○: The cells formed aggregates in three dimensions. Δ: The cells had a mixture of aggregates formed in three dimensions and the state of extending on the substrate. ×: Three-dimensional aggregates of cells were observed. Didn't exist

Claims (7)

  A method for culturing neural stem cells, comprising culturing cells containing neural stem cells on a fluoropolymer base material to form neurospheres retained on the base material.   The culture method according to claim 1, wherein the fluoropolymer contains an aromatic fluorine compound as a structural unit.   The culture method according to claim 1 or 2, wherein the culture is performed in the presence of one or more growth factors selected from the group consisting of EGF, bFGF, HGF, and LIF.   The culture method according to any one of claims 1 to 3, wherein the culture is performed in a serum-free medium. A neuron comprising: forming a neurosphere by the culture method according to any one of claims 1 to 4; and inducing differentiation of the neural stem cell in a state where the neurosphere is held on the base material. Spheroid formation method.   The neural stem cell differentiation induction is selected from the group consisting of serum, B27-supplement, SM-Neuronal supplement, retinoic acid and derivatives thereof, NGF, LIF, GDNF, CNTF, BDNF, NT-3, BMP2, BMP4, and SHH The formation method of Claim 5 performed by culture | cultivation with the culture medium containing 1 or more types.   The formation method according to claim 5 or 6, wherein the neurospheroid includes nerve cells.