JP6912789B2 - Neural stem cell culture method and neurospheroid formation method - Google Patents

Description

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

The cells that form each organ such as the liver, pancreas, skin, and blood vessels express their functions by forming a three-dimensional network of cells in the living body. Therefore, in research on regenerative medicine that regenerates the functions of these organs, when culturing cells in these organs, a culture that allows the cells to form a three-dimensional network (that is, three-dimensional culture) is required. Be done. When culturing cells on the surface of a general resin cell culture substrate, it is difficult for the cells to spread in a plane and proliferate (two-dimensional culture) to form a three-dimensional network.

Neural stem cells are cells that have the ability to differentiate into nerve cells and glial cells (for example, astrocytes, oligodendrocytes, etc.) and retain the ability to proliferate (self-renewal ability). By suspending neural stem cells, it is possible to form a three-dimensional network, either alone or together with other cells, to form cell aggregates called neurospheres. In the case of neural stem cells, when the cells are cultured on the surface of a general resin cell culture substrate, differentiation is induced and progressed by adhesion to the substrate. Therefore, in order to proliferate and maintain neural stem cells in an undifferentiated state to form neurospheres, it is necessary to avoid adhesion to the substrate, and conventionally, neurospheres have been formed by suspension culture. (Conventional "neural sphere method"). By inducing the differentiation of neural stem cells using the formed neurospheres, neurospheroids, which are cell aggregates containing nerve cells and glial cells, can also be formed. Neurospheres containing undifferentiated and proliferative neural stem cells and neurospheroids with a three-dimensional network similar to living organisms are used to study and treat nervous system diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. It will be a very useful tool in the development of law.

For the above reasons, in the development of cell culture vessels used for the formation of neurospheres, measures have been taken to reduce the adhesion of neural stem cells to the surface of the culture vessel. For example, as a technique for reducing the adhesion of neural stem cells to the surface of a culture vessel, Patent Document 1 describes a method for producing a neural stem cell agglomerate forming container, in which a water-soluble resin is coated on the inner surface of the container to make it water-soluble. It is characterized by including a water-soluble resin coating step of forming a resin coating layer, and a water-insoluble cured film modification step of curing the water-soluble resin coating layer to form a water-insoluble cured film layer after the step. An invention relating to a method for producing a container for forming a neural stem cell agglomerate is disclosed.

On the other hand, after induction of differentiation, it is not necessary to proliferate neural stem cells in an undifferentiated state, and it is not necessary to dare to culture in a floating system. Therefore, from the viewpoint of operability, it is common to culture neurospheroids in a state of being fixed on a substrate. Therefore, when forming neurospheroids, neural stem cells are cultured in a culture vessel having low cell adhesion as described above, and then the formed neurospheres are transferred to a culture vessel having high cell adhesion. A method of inducing differentiation has been adopted. However, inducing differentiation using a culture vessel different from the culture vessel used for forming neurospheres not only complicates the work process, but also causes the formed neurospheres to disintegrate during medium exchange or media. At the same time, some parts were lost. To solve such a problem, as a technique capable of forming a neurosphere and forming a neurospheroid in a single culture medium, for example, Patent Document 2 has a group having a predetermined chemical structure in the vicinity of the surface. A step of suspending and culturing nerve stem cells in a medium containing a growth factor to form a cell aggregate, and adding a differentiation-inducing factor to the medium to adhere the cell aggregate to the culture vessel. Described is an invention relating to a culture method for forming nerve cells and / or glial cells from nerve stem cells, which comprises a step of allowing the nerve stem cells to differentiate.

Japanese Unexamined Patent Publication No. 2008-220205 Japanese Unexamined Patent Publication No. 2013-135640

However, in the culture method described in Patent Document 2, although it is possible to obtain neural stem cells to post-differentiated nerve cells and / or glial cells using a single culture vessel, neurosphere formation can be performed. Must be performed in suspension culture. Culturing in a floating system still has problems such as neurosphere collapse and loss. In addition, when the neurospheres were cultured in a floating system, excessively large neurospheres were 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 inducing differentiation into neural stem cells, it is necessary to once collect the formed neurospheres and then perform the differentiation-inducing treatment, which complicates handling.

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 suspension culture of neural stem cells.

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

The present inventors have conducted diligent research in order to solve the above problems. As a result, it was found that the above-mentioned problems could be solved by culturing neural stem cells on a fluoropolymer-containing polymer substrate, and the present invention was 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 forming neurospheroids in a single cell culture vessel.

FIG. 1 is an optical microscopic image obtained by culturing neural stem cells on a fluoropolymer base material in Example 1 and confirming that neurospheres retained on the base material are formed (FIG. 1 (a) culture). 1st day, (b) 3rd day of culture, (c) 5th day of culture, (d) 8th day of culture). In FIG. 1, the upper row of each of (a) to (d) has a magnification of 40 times, the lower row has a magnification of 200 times, and the white bar has a magnification of 200 μm. FIG. 2 is an optical microscope image in which it was confirmed that neurospheroids were formed by subjecting the nerve stem cells cultured on the fluoropolymer substrate of Example 1 to differentiation induction treatment (FIG. 2 (a) differentiation medium). On the 0th day of the culture medium exchange, the 1st day of culturing in the differentiation medium, (b) the 3rd day of culturing in the differentiation medium, (c) the 5th day of culturing in the differentiation medium, and (d) the differentiation medium. 9th day of culture). In FIG. 2, the upper row of each of (a) to (d) has a magnification of 40 times, the lower row has a magnification of 200 times, and the white bar has a magnification of 200 μm. FIG. 3 is an optical microscope image of the neurospheroids formed by the suspension culture in Comparative Example 1 (FIG. 3 (a) culture day 1, (b) culture day 3, (c) culture day 5 and (FIG. 3). d) 8th day of culture). In FIG. 3, the upper row of each of (a) to (d) has a magnification of 40 times, the lower row has a magnification of 200 times, and the white bar has a magnification of 200 μm. FIG. 4 is an optical microscope image of the neurospheroids formed in the suspension culture in Comparative Example 1 when differentiation-inducing treatment is performed (FIG. 4 (a), the day of medium exchange to the differentiation medium is set as the 0th day). 1st day of culturing in the differentiation medium, (b) 3rd day of culturing in the differentiation medium, (c) 5th day of culturing in the differentiation medium, (d) 9th day of culturing in the differentiation medium). In FIG. 4, the upper row of each of (a) to (d) has a magnification of 40 times, the lower row has a magnification of 200 times, and the white bar has a magnification of 200 μm. FIG. 5 shows the results of measuring the expression levels of undifferentiated markers and differentiation markers expressed in cells before and after induction of differentiation in Example 1 and Comparative Example 1, respectively.

One aspect of the present invention relates to a method for culturing neural stem cells, which comprises culturing cells containing neural stem cells on a fluoropolymer base material to form neural stem cells retained on the base material. The present invention is based on the surprising finding that by culturing neural stem cells on a fluoropolymer-containing polymer substrate, neural stem cells can be proliferated while being retained on the substrate in an undifferentiated state, and neurospheres can be formed. It is a thing. According to the present invention, since it is not necessary to form neurospheres by suspension culture, the risk that the formed neurospheres disintegrate during medium exchange or a part of the formed neurospheres is lost together with the medium can be significantly reduced. Can be done. Further, according to the present invention, it is possible to effectively prevent the formation of excessively large neurospheres due to the association of neurospheres floating in the medium. The detailed mechanism by which neural stem cells, which could not be formed while being held on a substrate, can be formed by culturing neural stem cells on a fluoropolymer substrate is unknown, but is as follows. The reason is guessed. That is, although there are many unclear points about the detailed mechanism by which neural stem cells adhere to the substrate to induce differentiation, the physical stimulus of contact of the cells with the substrate is involved as an input of the differentiation induction signal. it is conceivable that. On the other hand, fluoropolymers are characterized by being hydrophobic and highly water repellent. Therefore, by culturing neural stem cells on a fluoropolymer base material, the hydrophobic nature of the fluoropolymer makes the interaction between the base material and the neural stem cells appropriate, and the differentiation-inducing signal is suppressed. It is speculated that it may be possible to retain the neurosphere on the material. The above mechanism is speculative and does not limit the technical scope of the present invention.

Another aspect of the present invention is that neural stem cells are cultured on a fluoropolymer base material to form a neurosphere held on the base material, and the nerve is held on the base material while holding the neurosphere. It relates to a method of forming a neurospheroid, including inducing the differentiation of stem cells. Such a technique capable of inducing differentiation of neurospheres held on a substrate enables the formation of neurospheres and neurospheroids in a single cell culture vessel. In addition, although the detailed mechanism is unknown, the present inventors have made the cells highly sensitive to the inducer when the induction treatment using the inducer is performed on the neurospheres retained on the fluoropolymer. They also found the advantage of responding and strongly inducing differentiation.

Hereinafter, embodiments of the present invention will be described. The present invention is not limited to the following embodiments.

In the present specification, "X to Y" indicating a range means "X or more and Y or less". Unless otherwise specified, operations and physical properties are measured under the conditions of room temperature (20 to 25 ° C.) / relative humidity of 40 to 50% RH.

<Neural stem cell culture method>
The present invention is characterized in that neural stem cells are cultured on a fluoropolymer base material. Examples of the fluorinated polymer include fluorinated polyimide, fluorinated polyaryletherketone, fluorinated polyester polyether, fluorinated polyether and the like. The fluoropolymer forming the base material may be a blend of two or more kinds of polymers having different chemical structures. The fluorine-containing polymer preferably contains an aromatic fluorine compound as a structural unit from the viewpoint of the strength and heat resistance of the obtained film. In one embodiment, the fluorinated polymer is preferably a fluorinated polyimide, more preferably a fluorinated 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, and is typically a polyamic acid obtained by polymerizing one or more kinds of acid dianhydride and diamine. 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 a part of the chemical structure. For the production of the fluorine-containing polyimide, a compound having a fluorine atom in the molecule is used as at least one compound of the acid dianhydride and the diamine.

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

The two-stage synthesis method of polyimide is a method of synthesizing polyamic acid as a precursor and converting polyamic acid into polyimide acid. The polyamic acid as a precursor may be a polyamic acid derivative. Examples of the polyamic acid derivative include a polyamic acid salt, a polyamic acid alkyl ester, a polyamic acid amide, a polyamic acid derivative from bismethylidene pyromeride, a polyamic acid silyl ester, and a polyamic acid isoimide.

As the one-step synthesis method of polyimide, a one-step synthesis method using a solvent such as a high-temperature melt polymerization method, an isocyanate method, a tetracarboxylic dianhydride method, and a method using an ionic liquid can be used. Other one-step synthesis methods include a polymerization method via a nylon salt-type 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.

Further, in the present invention, the "acid dianhydride residue" may be a tetravalent organic group forming the above structure, and does not have to be a residue actually formed by the reaction of the acid dianhydride. Similarly, the "diamine compound residue" may be a divalent organic group forming the above structure, and does not have to be a residue actually formed by the reaction of the 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 in which each of the two benzene rings of the biphenyl group is substituted with one amino group, and each amino group is substituted with a single bond to a nitrogen atom. Has a structure. ]
Includes repeating units indicated by.

In the present invention, a fluorine-containing polyimide containing a structural unit represented by the following formula (II) is preferable from the viewpoint of neurosphere forming property.

In formula (II) above, X represents either 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 independently represent either 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 Z 5 and At ^{least one of Z 6} contains one or more hydrogen atoms, and p is 0 or 1. In the fluorine-containing polyimide, the chemical structure represented by the formula (II) may be different or the same for each structural unit of the polymer.

In the above formula (II), when p = 0, X may not exist (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 arylentio group and the like, and among these, an alkylene group, an aryleneoxy group and an arylenthio group are included. 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 the total hydrogen atom in the divalent organic group is substituted with a fluorine atom. The alkylene group has, for example, 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms.

Examples of the alkylene group substituted with a fluorine atom, which is an example of X, include −C (CF ₃ ) ₂ −, −C (CF ₃ ) ₂ −C (CF ₃ ) _2−, and the like. Among the above-mentioned alkylene groups which are examples of X, −C (CF ₃ ) ₂₋ is preferable.

Examples of the arylene group as an example of X include the following.

Examples of the arylene oxy group, which is an example of X, include the following.

Examples of the arylentio group as an example of X include the following.

The above-mentioned allylene group, allyleneoxy group and allylenethio group which are examples of X are independently halogen atoms (for example, fluorine atom, chlorine atom, bromine atom, iodine atom, preferably fluorine atom or chlorine atom. , More preferably a fluorine atom.), May be substituted with a group selected from the group consisting of a methyl group and a trifluoromethyl group. The number of these substituents may be plural, and in that case, the types of the substituents may be the same or different from each other. Suitable substituents substituted with an arylene group, an aryleneoxy group and an arylentio group are a fluorine atom and / or a trifluoromethyl group, preferably a fluorine atom. The arylene group, the aryleneoxy group and the arylentio 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 an alkylene group having 1 to 4 carbon atoms having a single bond of two or more benzene rings (for example, methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, Isobutylene groups), groups having a structure that is directly bonded via or directly attached to an oxygen atom or a sulfur atom. Preferably, the divalent organic group represented by Y has a structure in which two or three benzene rings, each of which may have a substituent independently, are bonded via a single bond, an oxygen atom, or a sulfur atom. It is a group having. More preferably, the divalent organic group represented by Y has a structure in which two or three benzene rings, each of which may have a substituent independently, are bonded via an oxygen atom or a sulfur atom. It is a group. Substituents of the benzene ring include halogen atoms (eg, fluorine atom, chlorine atom, bromine atom, iodine atom, preferably fluorine atom or chlorine atom, more preferably fluorine atom), methyl group and trifluoro. Examples thereof 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 above-mentioned divalent organic group having an aromatic ring, which is an example of Y, is a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, preferably a fluorine atom or a chlorine atom, if substitutable. , More preferably a fluorine atom.), May be substituted with a group selected from the group consisting of a methyl group and a trifluoromethyl group. The number of these substituents may be plural, and in that case, the types of the substituents may be the same or different from each other. A suitable 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 have a structure represented by the following formula (III).

In formula (III), B ¹ represents CF ₃ or CN. B ² represents H, F, Cl, Br or I, the same or different. R ¹ represents a halogen-substituted alkyl group having 1 to 20 carbon atoms. X ^A represents O or S, which is the same or different. X ^B represents O or S. n represents the number of substitutions of the group represented by ^{B 2, and is an integer of 0 to 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 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 halogen atoms. However, the structure is not particularly limited, and may be any of a linear, branched, and cyclic alkyl group structure. Further, it may have an ether bond in the halogen-substituted alkyl group. 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. The carbon number of R ¹ is more preferably 2 to 18, and even more preferably 3 to 15.

Particularly suitable groups for R ¹ include, for example, 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 preferably O or both are preferably S, and most preferably both are O.

In formula (III), B ² represents H, F, Cl, Br or I in the same or different manner, ^{but it is preferable that at least one of B 2} is a halogen atom (F, Cl, Br or I). Is. Above all, it is preferable that the ^two B 2s in the formula (III) are both halogen atoms. Further, among the halogen atoms, a chlorine atom (Cl) and a fluorine atom (F) are preferable, and a fluorine atom (F) is more preferable. Particularly preferably, two B ² in formula (III) is, and that both a fluorine atom (F), forms thus the B ² are F (fluorine atom), also of the present invention It is one of the preferred embodiments.

From the viewpoint of neurosphere retention, 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 formula (III). The structure is preferably selected from the group, more preferably the structure selected from the group consisting of d-3 and e-1, and further preferably the 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 from each other, and each of them independently has a hydrogen atom and a fluorine atom. , Chlorine, bromine or iodine, and if at least one of X and Y does not contain a fluorine atom, then at least one of Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ^6. Is a fluorine atom. The number of fluorine atoms contained in the formula (II) may be one or more, but is preferably four 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 the viewpoint of retention of neurosphere, in one 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, an aryleneoxy group, and an arylentio group which may be substituted with a fluorine atom; Z ¹ , Z ² , Z. ³ , Z ⁴ , Z ⁵ , and Z ⁶ are similar to formula (II) above; Z ⁷ , Z ⁸ , Z ⁹ , Z ¹⁰ , Z ^11, and Z ¹² are independent of each other hydrogen atom, fluorine atom, Indicates either a chlorine atom, a bromine atom, an iodine atom, a methyl group or 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 contain one or more fluorine atoms; Y ¹ is independently selected from oxygen and sulfur atoms, respectively; q is an integer of 1-3, preferably 1. Or 2. Specific examples of X in formula (II-2) are as described above for formula (II).

More specifically, the structural unit represented by the above formula (II) is selected from the structural units represented by the following formulas (II-3) (6FDA / TPEQ) and (II-4) (6FDA / ODA). It is more preferable that it is one.

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

The polyamic acid synthesis reaction is preferably carried out 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 acid dianhydride as the raw material 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. , Polar solvents such as methanol; non-polar solvents such as toluene and xylene. Above all, 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 imidization. The concentration of the polyamic acid in the polyamic acid solution is not particularly limited, but is preferably 5 in view of the polymerization reactivity of the obtained resin composition, the viscosity after polymerization, and the ease of handling in subsequent film formation and firing. 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 (thermally imidized) by heat treatment to obtain a resin composition containing a fluorine-containing polyimide. The polyimide obtained by thermal imidization has no possibility of residual catalyst and is more preferable for cell culture applications. The imidization rate of the fluorine-containing polyimide resin does not have to be 100%. That is, the fluorine-containing polyimide resin partially contains a structural unit which is a ring-opened amide structure in a part of the cyclic imide structure of the structural unit represented by the above formula (I) or formula (II). You may.

When imidizing by thermal imidization, for example, the polyamic acid is placed in air, more preferably in an atmosphere of an inert gas such as nitrogen, helium, or argon, or in vacuum, preferably at a temperature of 50 to 400 ° C. , More preferably 100 to 380 ° C., preferably 0.1 to 10 hours, more preferably 0.2 to 5 hours, to carry out an imidization reaction to obtain a resin composition containing polyimide. be able to.

The polyamic acid to be subjected to the thermal imidization reaction is preferably in a form dissolved in a suitable solvent. The solvent may be any one that dissolves polyamic acid, and the above-mentioned solvent 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 in a suitable solvent by using the dehydration cyclization reagent described later.

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

Examples of the tertiary amine compound include trimethylamine, triethylamine, tripropylamine, tributylamine, pyridine, 1,4-diazabicyclo [2.2.2] octane (DABCO), and 1,8-diazabicyclo [5.4. 0] Undec-7-ene, 1,5-diazabicyclo [4.3.0] Nona-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 can be mentioned. Among these, pyridine, DABCO, N, N, N', N'-tetramethyldiaminomethane are preferable, and DABCO is more preferable. The tertiary amine may be only one kind or two or more kinds.

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. The carboxylic acid anhydride may be only one kind or two or more kinds.

As the solvent for dissolving the polyamic acid in chemical imidization, a polar solvent having excellent solubility is preferable. For example, tetrahydrofuran, N, N-dimethylacetamide, N, N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide and the like can be mentioned, and among these, N, N-dimethylacetamide, N, N-dimethylformamide and N are particularly mentioned. -It is preferable that at least one selected from the group consisting of methylpyrrolidone is used from the viewpoint of performing a uniform reaction. When these solvents are used as the solvent for the amidation reaction, the polyamic acid can be used as it is for chemical imidization without being separated from the reaction mixture after the amidation reaction (fluorinated polyaryletherketone).
Examples of the fluorinated polyaryletherketone include a fluorinated polyaryletherketone composed of a structural unit represented by the following formula (IV).

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

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

Is one of. ) Is the group represented by. ]
It is a fluorinated polyaryletherketone represented by.

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

In the present invention, the method for producing the fluorinated polyaryletherketone represented by the above formula (IV) will be described in detail, but from this description, the terminal of the fluorinated polyaryletherketone represented by the formula (IV) is , The benzene ring side containing a fluorine atom is fluorine, and the ^R41 side is a hydrogen atom. That is, the fluorinated polyaryletherketone represented by the formula (IV) is represented by the following formula (IV-3):

[In the formula, n represents the degree of polymerization, m is an integer of 0 or 1, and R ⁴¹ is as defined above. ]
It is considered to be a fluorinated polyaryletherketone represented by.

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

[In the formula, n represents the degree of polymerization. ]
It becomes a fluorinated polyaryletherketone indicated by.

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

[In the formula, n represents the degree of polymerization. ]
It becomes a fluorinated polyaryletherketone indicated by.

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

[In the formula, n represents the degree of polymerization, and R ⁴² is as described above. ]
It becomes a fluorinated polyaryletherketone indicated 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) has the following formula (IV-7):

[In the formula, R ⁴² is as described above. ]
It is a polymer containing a repeating unit represented by.

In a particularly preferred embodiment, the fluorinated polyaryletherketone represented by the formula (IV) is represented by the following formula (IV-8):

It is a polymer containing a repeating unit represented by.

Of these, the fluorinated polyaryletherketones represented by the formulas (IV-2) and (IV-4) are prepared in an organic solvent in the presence of a basic compound, and have the following formula (IV-9):

[In the formula, q is an integer of 0 or 1. ]
It is obtained by heating the 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.

Further, the fluorinated polyaryletherketone represented by the above (IV-6) can be obtained in the presence of a basic compound in an organic solvent according to the following formula (IV-10):

4,4'-bis (2,3,4,5,6-pentafluorobenzoyl) diphenyl ether represented by and the following formula (IV-11):

[In the formula, R ⁴² has the following structure:

Is one of. ]
It is obtained by heating the divalent phenol compound represented by.

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. The polymerization time varies depending on other reaction conditions, raw materials used, and the like, but is preferably 1 to 48 hours, more preferably 2 to 24 hours. Further, the polymerization reaction may be carried out under normal pressure or reduced pressure, but it is desirable to carry out the polymerization reaction under normal pressure from the viewpoint of equipment.

Examples of the organic solvent used in the above 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 pentafluorobenzoyldiphenyl 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 early stages of the reaction, the water by-product of phenoxide formation can be removed as an azeotropic product of toluene, regardless of the polymerization solvent.

The basic compound used in the present invention acts to promote the polycondensation reaction by collecting hydrogen fluoride generated by the polycondensation reaction, and further, in the case of the polycondensation reaction with a divalent phenol compound, it acts. , Has the effect of converting phenolic compounds into more reactive anions.

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

Further, in the present invention, the amount of the basic compound used is 0.5 with respect to 1 mol of the pentafluorobenzoyldiphenyl ether compound used in the case of the polymers of the formulas (IV-2) and (IV-4). It is 10 mol, preferably 0.5-5 mol, or in the case of the polymer of formula (IV-5), 1-20 mol, preferably 1-20 mol, relative to 1 mol of the pentafluorobenzoyldiphenyl ether compound used. Is 1-10 mol.

Examples of the divalent phenol compound used in the present invention include 2,2-bis (4-vidroxyphenyl) -1,1,1,3,3,3-hexafluoropropane (hereinafter, "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, based on 1 mol of 4,4'-bis (2,3,4,5,6-pentafluorobenzoyl) diphenyl ether. It is 9.9 to 1.1 mol.

For example, in the presence of a basic compound, 4,4'-bis (2,3,4,5,6-pentafluorobenzoyl) diphenyl ether is reacted with 6FBA as a divalent phenol compound in an organic solvent. Thereby, 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 if necessary, the distillate is washed to obtain a desired polymer. Alternatively, the polymer may be obtained by adding the reaction solution to a solvent having a low solubility of the polymer to precipitate the polymer as a solid, and separating the precipitate by filtration.

(Fluorinated polyester polyether)
Examples of the fluorinated polyester polyether include a fluorinated polyester polyether composed of a structural unit represented by the following formula (V).

[In the formula (V), m'and n'represent the number of fluorine atoms added to the benzene ring, which are the same or different, and are integers of 1 to 4. R ⁵¹ and R ⁵² represent divalent organic groups having 1 to 150 carbon atoms, which are the same or different from each other. p represents the degree of polymerization. ]
It is a fluorinated polyester polyether that requires 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 the above formula (V) may be used. 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, and when it is composed of different repeating units, it may be block-shaped. It may be in any form such as random.

In the repeating unit represented by the above formula (V), ^{the portion (-O-R 52-} O-) is any of the ortho-position, the meta-position, and the para-position with respect to the carbon having the ester bond of the benzene ring. Although it may be bonded to the carbon of the above, those bonded to the ortho-position or the para-position are preferable. In the fluorinated polyester polyether used in the present invention, the benzene ring having a fluorine atom is in a form in which a part or all of the four hydrogen atoms are replaced with a fluorine atom, and the hydrogen atom of the benzene ring is also used. May be in the form of being substituted with a halogen atom other than a fluorine atom or another substituent such as a substituent having an alkyl chain. Therefore, the total of hydrogen atom, fluorine atom, halogen atom other than fluorine atom, and other substituents in one benzene ring is 4.

R ⁵¹ and R ⁵² represent the same or different divalent organic groups 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 of the 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 represent substituents, which are the same or different, and one benzene ring. has a 0-4 ^{Y 1,} Y 2, ^{Y 3} and ^Y 4, 0 to 3 substituents the ^{Y 1} 'and ^{Y 2'.} Examples of the substituent represented by Y ¹ , Y ¹ ', Y ² , Y ² ', Y ³ and Y ⁴ include a halogen atom, an alkyl group which may have a substituent, an alkoxy group, and an alkylamino. Examples thereof include a group, an alkylthio group, an aryl group, an aryloxy group, an arylamino group, an arylthio group and the like.

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

In the fluorinated polyesters polyethers used in the present invention, in the above formula (V), the structure of R ⁵¹ is any of the above-described (9-6) or (9-18), and the benzene ring substituents It is preferable that the substance does not have. That is, the fluorinated polyester polyether has the following formula (V-2):

[In the formula, m'and n'represent the number of fluorine atoms added to the benzene ring, which are the same or different, and are integers of 1 to 4. R ⁵² represents a divalent organic group having 1 to 150 carbon atoms, which is the same or different. p represents the degree of polymerization. ]
Repeat unit represented by and / or the following formula (V-3):

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

The method for producing the fluorinated polyester polyether represented by the above formula (V) is not particularly limited, but a production method including a step of polymerizing the fluorinated ester compound and the dihydroxy compound is preferable. Further, 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) has the following formula (V-4):

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

In the formula, R ⁵² represents a divalent organic group having 1 to 150 carbon atoms. ]
It is preferably produced by a production method including a step of polymerizing the dihydroxy compound represented by (1) in the presence of a basic catalyst.

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, it is a uniform system. Various polymerization methods can be used regardless of the polymerization method, such as polymerization at 150 ° C. and interfacial polymerization, and polymerization is carried out even under conditions of 150 ° C. or lower, which is lower than the conventional polymerization reaction using a fluorine-containing compound. It is possible.

In the above production method, the structure (-O-R ^52- O-) derived from the dihydroxy compound is at any of the ortho-position, meta-position, and para-position with respect to the carbon to which the ester group of the benzene ring is bonded. It may be bonded to carbon, but it is preferably bonded to the ortho-position or the para-position. Further, when two or more portions derived from a dihydroxy compound are bonded to one benzene ring, a crosslinked structure may be formed. However, if the benzene ring has a crosslinked structure, the resulting polymer gels, so that the crosslinked structure is formed. The one with less is preferable. In the above production method, the ease of forming a crosslinked structure differs depending on the reaction temperature and reaction time, the type and concentration of the solvent and basic catalyst used, the order in which the raw materials are charged, the amount of water in the reaction system, etc. By making it possible, 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 of 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 fluorine-containing ester compound. More preferably, 0.9 to 1.1 mol of the 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. Further, the above reaction may be carried out under reduced pressure, normal pressure or pressurized, but it is preferable to carry out the reaction under normal pressure in consideration of equipment aspects and the like.

In the polycondensation reaction in the above production method, various solvents can be used due to the excellent solubility of the fluorine-containing ester compound in the solvent, and nitriles such as acetonitrile and benzonitrile; nitrobenzene and nitromethane. And other nitros; ketones such as acetone, methyl isobutyl ketone (MIBK), methyl ethyl ketone (MEK), cyclohexanone; halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, chloroethane, dichloroethane, trichloroethane, tetrachloroethane; Aromatic hydrocarbons such as benzene, toluene and xylene; hydrocarbons such as pentane, hexane, cyclohexane and heptane; ethers such as diethyl ether, isopropyl ether, tetrahydrofuran (THF), dioxane, diphenyl ether, benzyl ether and tert-butyl ether Types: Esters such as methyl formate, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate; N-methyl-2-pyrrolidinone (NMP), dimethylformamide (DMF), dimethylsulfoxide (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 any amount as long as the above reaction can proceed efficiently, but it is preferable that the amount of the fluorine-containing ester compound is 1 to 50% by mass in the solvent, and more preferably 1 to 30. The amount is such that mass% is present.

As the basic compound used in the polycondensation reaction in the above-mentioned production method, it acts to promote the polycondensation reaction by collecting hydrogen fluoride produced by the polycondensation reaction, and further, a dihydroxy compound as described above is used. Those having an action of converting into a highly reactive anion are preferable, for example, one of calcium carbonate, calcium hydroxide, potassium fluoride, tributylamine, pyridine, potassium carbonate, lithium carbonate, potassium hydroxide, triethylamine and the like. Two or more are suitable. The amount of such a basic compound used is preferably 0.5 to 20 mol with respect to 1 mol of the fluorine-containing ester compound used. More preferably, it is 0.8 to 10 mol.

After the 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, so that the fluorinated polyester polyether having the repeating unit represented by the above formula (V) is obtained. Will be obtained. It can also be obtained by adding the reaction solution to a solvent having a low solubility of the polymer to precipitate the fluorinated polyester polyether as a solid, and separating the precipitate by filtration.
(Fluorinated polyether)
Examples of the fluorinated polyether include a fluorinated polyether composed of a structural unit represented by the following formula (VI).

[In the formula (VI), R ³¹ contains 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. It may have an alkylamino group having 1 to 12 carbon atoms, an alkylthio group having 1 to 12 carbon atoms which may have a substituent, and an aryl having 6 to 20 carbon atoms which may have a substituent. A group, an aryloxy group having 6 to 20 carbon atoms which may have a substituent, an arylamino group which may have a substituent and may have 6 to 20 carbon atoms, or a carbon atom which may have a substituent. It represents the number 6-20 arylthio ^{group; R 61} represents a divalent organic group; and n represents a polymerization degree. ]
It is a fluorinated polyether represented 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; having 1-12 carbon atoms which may have substituents. Alkoxy groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentyloxy, hexyloxy, 2-ethylhexyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, furfuryloxy and allyloxy, preferably methoxy. , Ethoxy, propoxy, isopropoxy and butoxy; alkylamino groups having 1 to 12 carbon atoms which may have substituents, such as methylamino, ethylamino, dimethylamino, diethylamino, propylamino, n-butylamino, sec-butylamino and tert-butylamino, preferably methylamino, ethylamino, dimethylamino and diethylamino; alkylthio groups having 1 to 12 carbon atoms which may have substituents, such as methylthio, ethylthio, propylthio and n. -Butylthio, sec-butylthio, tert-butylthio and iso-propylthio, preferably methylthio, ethylthio and propylthio; aryl groups having 6 to 20 carbon atoms which may have substituents, such as phenyl, benzyl, phenethyl, o-, m- or p-tolyl, 2,3- or 2,4-xysilyl, mesityl, naphthyl, anthryl, phenanthryl, biphenylyl, benzhydryl, trityl and pyrenyl, preferably phenyl and o-, m- and p-tolyl An aryloxy group having 6 to 20 carbon atoms which may have a substituent, for example, phenoxy, benzyloxy, hydroxybenzoic acid and its esters (for example, methyl ester, ethyl ester, methoxyethyl ester, ethoxyethyl ester). , Flufuryl ester, phenyl ester, etc .; the same applies hereinafter), naphthoxy, o-, m- or p-methylphenoxy, o-, m- or p-phenylphenoxy, phenylethynylphenoxy, and cresotinic acid. And groups derived from esters thereof, preferably phenoxy and naphthoxy; arylamino groups having 6 to 20 carbon atoms which may have substituents, 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. It represents an arylthio group having 6 to 20 carbon atoms, for example, phenylthio, phenylmethanethio, o-, m- or p-tolylthio, and a group derived from thiosalicylic acid and its esters, preferably phenylthio. Of these, an optionally substituted aryloxy group is preferably an arylthio group, and an arylamino group, further, phenoxy, phenylthio and anilino is most preferred as R ^31.

Further, in the above formula (VI), a ^{substituent that can be used when R 31} represents an alkyl group having a substituent, an alkoxy group, an alkylamino group, an alkylthio group, an aryl group, an aryloxy group, an arylamino group or an arylthio group. Can be appropriately selected depending on the desired properties of the target product, and is not particularly limited, but for example, an alkyl group having 1 to 12 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl. , Se-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 The group and the like can be mentioned. 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:

The divalent organic group represented by is ^{preferable as R 61} , and in particular, the following formula:

The divalent organic group represented by is preferable as ^{R 61.}

Further, in the above formula (VI), n represents the degree of polymerization, specifically 5 to 1000, preferably 10 to 500. The fluorinated polyether used in the present invention may be composed of the same repeating unit or different repeating units of the constituent units of the above formula (VI), and in the latter case, it may be composed of different repeating units. The repeating unit may be either block-shaped or random-shaped.

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

It is considered to be the polymer indicated by.

The fluorinated polyether is represented by the following formula (VI-2):

The tetrafluorobenzonitrile derivative represented by the following formula (VI-3):

It is produced by polymerizing with the dihydroxy compound represented by (1) in the presence of a basic catalyst. ^{At this time, the definitions of R 31} in the above formula (VI-2) ^{and R 61 in} the above formula (VI-3) are the same as the definitions 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, and for example, the formula: R ³¹ H [in the formula, R ³¹ is the same as the definition in the above formula (VI). Is obtained by reacting the compound represented by [is] with 2,3,4,5,6-pentafluorobenzonitrile (also referred to as "PFBN" in the present specification) in the presence of a basic compound in an organic solvent. Be 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 in the form of a mixture of ^{two or more compounds represented by the formula: R 31 H and / or PFBN.} However, it is preferable to use it as a single compound in consideration of the purification process and the physical characteristics of the polymer. In the latter case, the total number of moles of the plurality or single PFBN used may be equal to or approximately equal to the total number of moles of the compound represented by the ^{multiple or single formula: R 31 H.} preferred, specifically, the ^formula: the amount of ^{R 31} compound represented by H is relative PFBN 1 mol, preferably 0.1 to 5 moles, more preferably 0.5 to 2 moles.

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 and the like. Examples thereof include a mixed solvent with a non-polar 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 PFBN in the organic solvent is 1 to 40% by mass, preferably 5 to 30% by mass. At this time, 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 azeotropic product of toluene regardless of the polymerization solvent.

Further, it is desirable that the basic compound used in the above reaction acts to collect hydrogen fluoride produced in order to promote the reaction. Examples of such a basic compound include potassium carbonate, calcium carbonate, potassium hydroxide, calcium hydroxide, potassium fluoride, triethylamine, tributylamine and pyridine. At this time, the amount of the basic compound used is 0.1 to 5 mol, preferably 0.5 to 2 mol, based on 1 mol of the PFBN used.

Further, 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, but for example, the reaction preferably puts the reaction system in a stirred state. While maintaining, it is usually carried out at a temperature of 20 to 180 ° C., preferably 40 to 160 ° C. The reaction time varies depending on other reaction conditions, raw materials used, and the like, but is usually 1 to 48 hours, preferably 2 to 24 hours. Further, the reaction may be carried out under normal pressure or reduced pressure, but it is desirable to carry out the reaction under normal pressure from the viewpoint of equipment. The product obtained by such a reaction is obtained by pouring distilled water into the reaction mixture, extracting with an extractant such as dichloromethane, dichloroethane or carbon tetrachloride, separating the organic layer from the extract, and retaining the extractant. Obtained by leaving. Further, the product may be obtained as crystals by recrystallization with methanol, ethanol or the like, if necessary.

The tetrafluorobenzonitrile derivative of the formula (VI-2) thus synthesized is further subjected to polymerization in the presence of the dihydroxy compound of the formula (VI-3) and a basic catalyst as described above. Produces a fluorinated polyether of the formula (VI) of interest. At this time, the tetrafluorobenzonitrile derivative of the formula (VI-2) may be used after undergoing purification steps such as extraction, recrystallization, chromatography and distillation as described above, or may be used as it is without any purification step. Although it may be used, it is preferable to use it after purification in consideration of the yield of the next step and the like.

The dihydroxy compound of the formula (VI-3) used in the above reaction is selected according to the structure of the fluorinated polyether of the formula (VI) which is the target product. Preferred dihydroxy compounds of the formula (VI-3) include 2,2-bis (4-vidroxyphenyl) -1,1,1,3,3,3-hexa as shown below. Fluoropropane (hereinafter referred to as "6FBA"), 4,4'-dihydroxydiphenyl ether (hereinafter referred to as "DPE"), hydroquinone (hereinafter referred to as "HQ"), bisphenol A (hereinafter referred to as "BA"), 9, 9-bis (4-hydroxyphenyl) fluorene (hereinafter referred to as "HF"), phenolphthaline (hereinafter referred to as "PP"), 9,9-bis (3-methyl-4-hydroxyphenyl) fluorene (hereinafter referred to as "PF"). "MHF"), 1,4-bis (hydroxyphenyl) cyclohexane (hereinafter "CHB"), and 4,4'-dihydroxybiphenyl (hereinafter "BP").

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

The above reaction may be carried out in an organic solvent or in the absence of a solvent, but is preferably carried out in an organic solvent. In the former case, the organic solvents that can be used 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 non-polar 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. At this time, 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 azeotropic product of toluene regardless of the polymerization solvent.

Further, in the present invention, it is essential that the reaction of the tetrafluorobenzonitrile derivative of the formula (VI-2) and the dihydroxy compound of the formula (VI-3) is carried out in the presence of a basic catalyst. The basic catalyst preferably has an action of converting the dihydroxy compound of the formula (VI-3) into a more reactive anion so as to promote the polycondensation reaction by the dihydroxy compound of the formula (VI-3). Examples include potassium carbonate, calcium carbonate, potassium hydroxide, calcium hydroxide, potassium fluoride and the like. 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).

Further, the reaction conditions in the above 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. The polymerization time varies depending on other reaction conditions, raw materials used, and the like, but is preferably 1 to 48 hours, more preferably 2 to 24 hours. Further, the polymerization reaction may be carried out under normal pressure or reduced pressure, but it is desirable to carry out the polymerization reaction under normal pressure from the viewpoint of equipment.

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

(Properties of fluoropolymer)
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 to 19% by mass or less. The fluorine content can be measured by the method described in Examples.

The cell culture surface of the base material 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. Less than °, preferably less than 120 °. The static water contact angle is a value measured by the method described in Examples.

The fluoropolymer used in the present invention preferably has a tensile elastic modulus of more than 2 GPa. By using a fluoropolymer having a tensile elastic modulus of 1 GPa or more, the retention of neurospheres is further improved. The tensile elastic modulus of the fluoropolymer 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 in reality, it is, for example, 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, the presence of many ether bonds in the polymer backbone can result in a flexible structure. Therefore, the tensile elastic modulus can be increased by reducing the number of groups that give flexibility to the molecule such as ether bond per molecule of the polymer by selecting a material or lowering the degree of polymerization. .. Alternatively, in the case of fluorine-containing polyimide, for example, the tensile elastic modulus can be controlled by appropriately setting the conditions for the imidization reaction (for example, the tensile elastic modulus can be increased by raising the firing temperature). Can be done. The tensile elastic modulus is a value measured by the method described in the examples.

The weight average molecular weight of the fluoropolymer is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, more preferably 10,000 to 500,000, and even more preferably. Is 100,000 or more and less than 300,000. In this specification, the weight average molecular weight of the polymer is a value measured by the method described in Examples.

The culture vessel used for culturing is not particularly limited as long as the cell adhesion surface is composed of a fluoropolymer base material. The cell culture container may be configured by combining a fluoropolymer base material and other members, or may be configured by integrating the fluoropolymer base material and other members. , It may be composed of only a fluoropolymer base material. When the fluoropolymer-containing polymer base material is a flexible base material such as a film, it may be formed in combination with an appropriate support member having rigidity. Examples of the material constituting the support member include inorganic glass such as soda glass, non-alkali glass, and boronic acid glass; carbon; metal such as silicon; polyolefin resin such as polyethylene, polypropylene, and cyclic olefin; polyethylene terephthalate (PET). Polypropylene resin such as; Acrylic resin such as polymethyl methacrylate; Epoxy resin; Polyvinyl chloride, Polyvinylene sulfide, Polystyrene resin, Polyvinyl acetate, ABS (Acrylonitrile-butadiene-styrene) resin, Polycarbonate resin, Vinyl ether, Polyacetal, etc. Examples thereof include polyphenylene ether (PPE), polyaryl ether, polyphenylene sulfide (PPS), polyether ether ketone (PEEK), polyaryl ether ketone, phenol resin, polyether nitrile (PEN), and quartz. The cell culture container may have any shape as a whole as long as the cell adhesion surface is provided with a fluoropolymer base material. For example, it can be in the form of a culture plate such as a single or multi-well plate, or various containers such as a petri dish, dish, flask, or bag. The cell culture container may also be in the form of a cell culture container in a culture device such as a mass culture device or a perfusion culture device.

(Neural stem cells)
As used herein, 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 has a proliferative ability (self-renewal ability). The 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. 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 and pigs. Examples thereof include cows, goats, sheep, horses, chickens, quail, mink, tupai, Xenopus laevis, and zebra fish. Neural stem cells may also be collected from living organisms, or may be pluripotent cells, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), or the like. The pluripotent cells may be induced to differentiate into neural stem cells by the method described in International Publication No. 2011/108766 and the like. Induction of differentiation of totipotent cells and pluripotent cells into neural stem cells may be carried out on the fluoropolymer base material according to the present invention.

When neural stem cells are recovered from living tissue and used, the tissue from which they are derived is not particularly limited and may be either central nervous system tissue or peripheral nervous system tissue, but for example, neural stem cells derived from the brain are preferably used. Can be done.

As used herein, 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. The size of the above neurospheres is an average value for 10 or more neurospheres observed with an optical microscope.

When neurospheres were formed by conventional suspension culture, excessively large neurospheres were sometimes formed due to the association of neurospheres floating in the medium. In this case, the cells present in the central part of the neurosphere may become necrotic. Therefore, in order to prevent the formation of an excessively large neurosphere, conventional 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 base material. That is, the presence of cells other than neural stem cells (for example, neural progenitor cells, glial blast cells, etc.) in the neurosphere is permitted to the extent that the intended effect of the present invention is not impaired. In the present invention, nerve stem cells have pluripotency such as cells other than nerve 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 and the like). Cells; fat cells, hepatocytes, kidney cells, pancreatic cells, mammary cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, myocardial cells, dendritic cells, chondrocytes, bones, including various precursor cells and stem cells Neurosphere formation in the presence of blast cells, osteoclasts, bone cells, fibroblasts, various bloodline cells, retinal cells, corneal-derived cells, gonad-derived cells, various stem cells; various cancer cells, etc. May be 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 the 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 the basal medium, Eagle's minimum essential medium (E-MEM), Dalveco modified Eagle medium (D-MEM), α-MEM, Glasgow MEM (G-MEM), IMDM, RPMI1640, HAM's F-10 medium ( Examples thereof include F-10), HAM's F-12 medium (F-12), MCDB medium, Williams medium E, Neurobasal medium, Natural Projector Basic medium, NS-A medium and the like. As the basal medium, a mixed medium such as D-MEM / F-12 in which the above-mentioned medium is mixed at an arbitrary ratio may be used. In addition, if necessary, growth factors, antibiotics, lipids, hormones (eg, insulin, dexamethasone, hydrocortisone, β-estradiol, progesterone, etc.), iron sources (eg, transferrin, etc.), organic acids (eg, pyruvate, etc.), 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 selenate, etc.), reducing agents. (For example, 2-mercaptoethanol, etc.), ethanolamine, serum protein (for example, albumin, etc.), buffer (for example, HEPES, etc.), various supplements for cell culture (for example, N2-supplement, ITS-supplement, GlutaMAX-I) You may use a medium to which an additive such as a supplement (such as a supplement) and a BMP inhibitor (for example, Noggin etc.) is added.

The growth factor added to the medium is not particularly limited as long as it can be used at a concentration that promotes the proliferation of neural stem cells and does not induce differentiation. From the viewpoint of cell proliferation, the culture of nerve stem cells is composed of a group consisting of EGF (epidermal growth factor), bFGF (basic fibroblast growth factor), HGF (hepatocyte growth factor), and LIF (leukemia inhibitor). It is preferably carried out in the presence of one or more of the growth factors of choice. The amount of these growth factors added may be appropriately adjusted depending on the cell density and 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, it is preferable to culture neural stem cells in a serum-free medium from the viewpoint that differentiation induction is effectively suppressed. In the present specification, the serum-free medium means a basal medium containing no prepared or unpurified serum, and a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors) is used. It shall correspond to a serum-free medium. In particular, from the viewpoint of proliferation, the medium used for culturing neural stem cells is preferably a serum-free medium containing an N2-supplement (Bottenstain, J. et al .: Methods Enzymol., 58, 94 (1979)). ..

Hereinafter, as a 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 (density of the entire cell including neural stem cells and other cells) at the start of culturing of neural stem cells on a fluoropolymer-containing polymer substrate 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 culture is carried out under the conditions usually used in the present technical field. For example, the culture conditions are about 25 to 40 ° C., preferably 32 to 38 ° C. under _{a CO 2} atmosphere of 1 to 10% (v / v), preferably about 5% (v / v), and the desired size. This is done until a neurosphere is formed (for example, about 3 to 15 days). The medium is preferably changed at a pace of once every 2 to 5 days. The medium exchange may be a full exchange or a partial exchange (for example, a half exchange).

The presence of undifferentiated neural stem cells in the formed cell aggregates can be confirmed by the expression of molecular markers of neural stem cell genes and proteins. As the molecular marker of such neural stem cells, conventionally known ones can be used, and for example, a plurality of molecular markers such as intermediate filaments such as nestin may be measured in combination as needed. The molecular marker may be measured by conventionally known means such as PCR (for example, quantitative PCR) or Northern blotting for genes, and ELISA method or immunostaining method for proteins.

<Method of forming neurospheroids>
One aspect of the present invention comprises forming a neural stem cell by the method for culturing a neural stem cell, and inducing differentiation of the neural stem cell while holding the neural stem cell on the substrate. A method of forming is provided. By forming a neurospheroid through the formation of a neurosphere, it is possible to obtain a neurospheroid in which a three-dimensional network is formed with an arbitrary size and shape. In particular, when the induction treatment using an inducer is performed on the neurosphere retained on the fluoropolymer, the cells respond with high sensitivity to the inducer, and differentiation can be strongly induced.

The neurospheroid is an aggregate formed by a plurality of cells including nerve cells, glial cells (for example, astrocytes, oligodendrocytes, etc.) and / or progenitor cells thereof. Neurospheroids are undifferentiated cells such as neural stem cells and 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. Pluripotent cells; including various precursor cells and stem cells, fat cells, hepatocellular cells, renal cells, pancreatic cells, mammary gland cells, endothelial cells, epithelial cells, smooth muscle cells, myoblasts, myocardial cells, dendritic cells Includes cells, cartilage cells, osteoblasts, osteoblasts, bone cells, fibroblasts, various blood lineage cells, retinal cells, corneal-derived cells, gonad-derived cells, various line cells; cells such as various cancer cells, etc.) You may be. Neurospheroids may contain either nerve cells or glial cells, or both. In one embodiment, a neurospheroid containing nerve cells is provided, and in another embodiment, a neurospheroid containing glial cells is provided. The shape and size of the neurospheroid are not particularly limited and can be appropriately set according to the intended use and purpose of use. For example, the diameter (excluding neurites) is 10 to 1000 μm, preferably 50 to 500 μm, and more preferably 100. It is ~ 200 μm. The size of the above neurospheroids is an average value for 10 or more neurospheroids observed with an optical microscope.

Neural stem cell differentiation induction is usually carried out 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 nerve stem cells is not particularly limited, but is, for example, Eagle's Small Essential Medium (E-MEM), Dalbecco Modified Eagle's Medium (D-MEM), α-MEM, and Glasgow MEM (G-MEM). MEM), IMDM, RPMI1640, HAM's F-10 medium (F-10), HAM's F-12 medium (F-12), MCDB medium, Williams medium E, Neurobasal medium, Natural Projector Basic medium, NS-A medium. And so on. As the basal medium, a mixed medium such as D-MEM / F-12 in which the above-mentioned medium is mixed at an arbitrary ratio may be used. In addition, if desired, growth factors (eg, EGF, FGF (eg, bFGF), IGF, HGF, TGF-β, etc.), antibiotics, lipids, hormones (eg, insulin, dexamethasone, hydrocortisone, β-estradiol, etc.), 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, glucose, heparin, etc.) , Ascorbic acid, biotin, etc.), salts (eg, sodium selenate, etc.), reducing agents (eg, 2-mercaptoethanol, etc.), ethanolamine, serum proteins (eg, albumin, etc.), buffers (eg, HEPES, etc.) ), Various supplements for cell culture (for example, N2-supplement, ITS-supplement, GlutaMAX-I supplement, etc.) may be added to the medium.

As the inducer (differentiation inducer), conventionally known inducers can be appropriately selected and used. More specifically, the induction of neurostem cell differentiation is described, for example, in serum (eg, animal serum such as bovine fetal 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-[[(5,5) 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 palmitate, etc.), NGF (nerve growth factor), LIF (prevention of leukemia) Factor), GDNF (Glia cell-derived neurotrophic factor), CNTF (hairy neurotrophic factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin-3), BMP2 (bone-forming protein-2) ), BMP4 (bone-forming protein-4), and SHH (Sonic Hedgehog) by culturing in a medium containing one or more selected from the group. The inducer (differentiation inducer) may be used alone or in combination of two or more, and the composition may be appropriately changed according to the degree of progress of differentiation. As a method for inducing differentiation into nerve cells, the differentiation induction of neural stem cells is cultured in a medium containing a B27-supplement (Brewer, GJ et al .: J. Neurosci. Res., 35,567 (1993)). It is more preferable to carry out by culturing in a Neurobasal medium containing a B27-supplement. Although the detailed mechanism is unknown, when a fluoropolymer is used as a base material, the responsiveness of the differentiation signal by the inducer is improved as compared with the case of suspension culture, and the differentiation signal is particularly improved by using B27-supplement. The responsiveness of is particularly significantly improved. The amount of B27-supplement added is, for example, 1 to 4% (v / v) in the medium.

The culture conditions are not particularly limited, and the culture is carried out under the conditions usually used in the present technical field. For example, the culture conditions are about 25 to 40 ° C., preferably 32 to 38 ° C. under _{a CO 2} atmosphere of 1 to 10% (v / v), preferably about 5% (v / v), and the desired size. This is done until a neurosphere is formed (for example, about 1 to 20 days). The medium is preferably changed once every 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 expression of molecular markers such as genes, proteins and sugars. As such a differentiation marker, conventionally known ones can be appropriately selected and used, and for example, PSA-NCAM (polysialic acid nerve adhesion molecule), β-tubulin-3, NeuN, GFAP (glial cell fibrous acidity) can be used. Molecular markers such as protein), PLP (proteolipid protein), and MBP (myelin basic protein) may be appropriately combined and measured as necessary. For the measurement of molecular markers, conventionally known means such as PCR (for example, quantitative PCR) and Northern blotting for genes and ELISA method and immunostaining method for proteins can be adopted.

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

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

(Measurement of weight average molecular weight)
Equipment: HCL-8220GPC manufactured by Tosoh Corporation
Column: TSKgel Super AWM-H
Eluent (LiBr · _H 2 0, phosphoric acid containing NMP): 0.01 mol / L
Measurement method: A 0.5% by weight 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)
Equipment: Automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500)
Measuring 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)
Device: Dynamic viscoelasticity (manufactured by TA Instruments: RSA G2)
Measuring method: A film having a thickness of 40 μm was cut into strips of 5 mm × 40 mm, elongation and stress at 25 ° C. were measured, and a tensile elastic modulus was calculated.

(Manufacturing of fluoropolymer base material)
2.976 g (10.2 mmol) of 1,4-bis (aminophenoxy) benzene (TPEQ) and 4.524 g (6FDA) of 4,4'-hexafluoroisopropyridene diphthalic anhydride (6FDA) in a 100 ml volumetric flask. 10.2 mmol), 42.5 g of N, N-dimethylacetamide was charged. A fluoropolyamic acid resin composition (solid content concentration: 15.0% by mass) was obtained by stirring at room temperature for 5 days in a nitrogen atmosphere. 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 fluorine-containing polyimide after firing are substantially the same.

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

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

(Culturing neural stem cells)
A 24-well plate placed on the cell culture surface using the fluorine-containing polyimide film 1 obtained above as a base material was prepared and used for culturing neural stem cells by the following method. The fluorine-containing polyimide film was sterilized and used for cell culture.

A 14-day-old fetus was removed from Wistar rats, and the brain was collected from the removed fetus. The recovered fetal brain was treated with 2 ml of a cell separation / dispersion solution (Accumax, manufactured by Innovative Cell Technologies) at room temperature for 10 to 15 minutes 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 through a filter having a pore size of 30 μm. The solution was centrifuged at 900 rpm for 5 minutes. After centrifugation, the supernatant is 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). MEM / F-12 (1: 1 (v: v)) in a medium (Sigma-Aldrich, Inc.)), and the precipitate resuspended to a concentration of 2.5 × 10 ⁵ cells / ml. ^{The cell suspension prepared above was seeded (5 × 10 4} cells / cm ² ) on a 24-well plate on which a fluoropolymer-containing polymer substrate was placed on the cell culture surface (day 0 of culture). Then, the cells were cultured in a 5% (v / v) CO ₂ incubator at 37 ° C., the medium was exchanged by half amount exchange on the 3rd day of the culture, and the cells were cultured until the 8th day. It was confirmed by an optical microscope that neurospheres retained on the substrate appeared with the progress of the culture. The observation image by the optical microscope is shown in FIG. 1 (FIG. 1 (a) culture day 1, (b) culture day 3, (c) culture day 5, (d) culture day 8). The size of the neurospheres on the 8th day of culture was 171 ± 66 μm in diameter (mean ± standard deviation μm of 41 neurospheres).

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

Using the recovered RNA as a sample, the gene expression levels of nestin (undifferentiated marker) and β-tubulin-3 (differentiation marker) were measured by a quantitative PCR method. As a kit for quantitative PCR, TaqMan Universal PCR Master Mix and TaqMan Gene Expression Expressions (manufactured by Applied Biosystems) were used, and the measuring device name was: 7300/7500 Realtime PCR Systems (manufactured by Applied Biosystems). The gene expression levels of Nestin and β-tubulin-3 were calibrated as their relative levels 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 were measured. Calculated as 1. The results are shown in FIG.

(Formation of neurospheroids)
After confirming the formation of neurospheres on the 8th day of culturing, the medium was replaced with a differentiation medium (Neurobasal medium (Life technologies) containing a 2% (v / v) B27-supplement (Life technologies)). The culture was continued while changing the medium by half every day, and it was confirmed by an optical microscope that neurospheroids were formed.

The observation image of the neurospheroids by an optical microscope is shown in FIG. , (C) 5th day of culturing in the differentiation medium, (d) 9th day of culturing in the differentiation medium). As shown in FIG. 2, when the fluoropolymer base material was used, a three-dimensional network structure in which neurospheroids were linked to each other was confirmed after the 5th day of culturing in the differentiation medium.

On the 11th day (19th day after neural stem cell seeding) after replacement with the differentiation medium, cells were collected using a cell separation / dispersion solution (Accumax, manufactured by Innovative Cell Technologies) in the same manner as above, and RNA was collected. Recovered.

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

<Comparative example 1>
Cell culture The neural stem cells were cultured by the same method as in Example 1 except that a suspension culture plate was used, in which the surface of the substrate was surface-treated to reduce the adhesion of cells to the substrate. Observation images of neural stem cells cultured using a 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) Shown in (8th day of culture). In the suspension culture system culture plate, the neurosphere was not retained on the substrate and was suspended in the medium. In addition, the size of the neurosphere was enormous as compared with the case of culturing on a fluoropolymer base material.

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

On the 8th day of culturing, culturing in a differentiation medium was started by the same method as in Example 1 except that a suspension culture plate was used instead of the plate using the fluoropolymer base material.

Observation images of nerve stem cells cultured in a differentiation medium using a suspension culture plate with an optical microscope are shown in FIG. 4 (FIG. 4A) Culture in a differentiation medium with the day of medium exchange to the differentiation medium as the 0th day. It is shown on the day, (b) the third day of culturing in the differentiation medium, (c) the fifth day of culturing in the differentiation medium, and (d) the ninth day of culturing in the differentiation medium). As shown in FIG. 4, when culturing in suspension culture, it was not possible to confirm the three-dimensional network structure in which neurospheroids were connected to each other.

On the 11th day (19th day after neural stem cell seeding) after replacement with the differentiation medium, cells were collected using a cell separation / dispersion solution (Accumax, manufactured by Innovative Cell Technologies) in the same manner as above, and RNA was collected. Recovered.

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

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

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

<Examples 2 and 3>
(Manufacturing of fluoropolymer base material)
2.330 g (11.64 mmol) of 4,4'-diaminodiphenyl ether (ODA) and 42.5 g of N, N-dimethylacetamide were charged and dissolved in a 100 ml volumetric flask. To this, 5.170 g (11.64 mmol) of 4,4'-hexafluoroisopropyridene diphthalic acid anhydride (6FDA) was added, and the mixture was stirred at room temperature for 5 days under a nitrogen atmosphere to obtain a fluorine-containing polyamic acid resin. A composition (solid content concentration 15.0% by 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 fluorine-containing 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 by the same method as in Example 1.

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

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 in a 100 ml volumetric flask. 4.359 g (9.81 mmol) of 4,4'-hexafluoroisopropyridene diphthalic acid anhydride (6FDA) was added thereto, and the mixture was stirred at room temperature for 5 days under a nitrogen atmosphere to obtain a fluorine-containing polyamic acid resin. A composition (solid content concentration: 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 fluorine-containing 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 by the same method as in Example 1.

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

(Culturing neural stem cells)
Using the fluorine-containing polyimide film 2 and the fluorine-containing polyimide film 3 obtained above as base materials, 24-well plates arranged on the cell culture surface were prepared, 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 agglomerates in three dimensions Δ: The cells had a mixture of a part in which the agglutinates were formed in three dimensions and a state in which the cells were extended on the substrate ×: Three-dimensional agglutinations of the cells were observed. Did not

Claims (6)

Including culturing cells containing neural stem cells on a fluoropolymer base material to form neurospheres retained on the base material.
The fluoropolymer has the following formula (II-2):

In the above formula (II-2), X is −C (CF ₃ ) ₂ − , Y ¹ is an oxygen atom , and 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} are water MotoHara child, q is 1 or the structural unit represented by is 2; or by the following formula (II):

In the above formula (II),
X is −C (CF ₃ ) _2-
Y has a structure represented by the following formula d-9.

Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ are hydrogen atoms.
A method for culturing undifferentiated neural stem cells, wherein p contains the structural unit indicated by 1. The culture method according to claim 1, wherein the culture is carried out 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 claim 1 or 2 , wherein the culture is carried out in a serum-free medium. A neuro that comprises forming a neurosphere by the culture method according to any one of claims 1 to 3 and inducing differentiation of the neural stem cell while holding the neurosphere on the substrate. How to form spheroids. The induction of neural stem cell differentiation is selected from the group consisting of serum, B27-supplement, SM-Neuronal supplement, retinoic acid and its derivatives, NGF, LIF, GDNF, CNTF, BDNF, NT-3, BMP2, BMP4, and SHH. The formation method according to claim 4 , wherein the formation method is carried out by culturing in a medium containing one or more of the above-mentioned species. The formation method according to claim 4 or 5 , wherein the neurospheroid comprises a nerve cell.

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