WO2013099506A1

WO2013099506A1 - Culture method for forming nerve cells and/or glial cells from neural stem cells

Info

Publication number: WO2013099506A1
Application number: PCT/JP2012/080790
Authority: WO
Inventors: 隆安田; 誠山中; 啓吾武井; 如光隅田
Original assignee: 株式会社資生堂
Priority date: 2011-12-28
Filing date: 2012-11-28
Publication date: 2013-07-04
Also published as: JP2013135640A

Abstract

One embodiment of the invention is a culture method for forming nerve cells and/or glial cells from neural stem cells, having: a step for suspension culturing neural stem cells in a medium containing a growth factor and forming a cell aggregate, using a culture vessel; and a step for adding a differentiation-inducing factor to the medium, adhering the cell aggregate to the culture vessel, and differentiating the neural stem cells. The culture vessel has groups represented by general formula (1) (in the formula, R¹, R², and R³ are each independently an alkyl group having from 1 to 6 carbon atoms; m is an integer of from 2 to 6) present near the surface.

Description

Culturing method for forming neural cells and / or glial cells from neural stem cells

One embodiment of the present invention relates to a culture method for forming neural cells and / or glial cells from neural stem cells.

Conventionally, as a method for inducing differentiation of neural stem cells into neural cells and / or glial cells, a method is known in which neural stem cells are cultured in suspension to form cell aggregates and then differentiated into neural cells and / or glial cells. .

In Patent Document 1, a first base material in which a through hole is formed, a second base material that is peelably bonded to the surface of one side of the first base material and covers one side of the through hole, A cell assembly forming instrument having a non-cell-adhesive well formed by covering one side with a second substrate is disclosed.

Patent Document 1 discloses a cell assembly transfer kit including a cell assembly forming instrument and a culture instrument having a cell adhesive surface that abuts the surface of the first base of the cell assembly forming instrument. It is disclosed.

Further, Patent Document 1 discloses an assembly forming step for forming a cell assembly in a floating state in a well of a cell assembly forming device, and a surface on the other side of the first base material of the cell assembly forming device. A transfer process in which the cell aggregate in the well is brought into contact with the cell adhesive surface to settle on the cell adhesive surface and the second substrate is peeled off from the first substrate, and the cells adhered to the cell adhesive surface A method for culturing a cell aggregate including an aggregate culture step for culturing the aggregate is disclosed.

However, there is a problem that without using a cell assembly formation device and a culture device together, nerve cells and / or glial cells cannot be formed from neural stem cells via cell aggregates.

JP 2010-233456 A

One embodiment of the present invention is capable of forming nerve cells and / or glial cells from neural stem cells via cell aggregates using a single culture vessel in view of the problems of the above-described conventional techniques. An object is to provide a culture method.

One embodiment of the present invention is a culturing method for forming neural cells and / or glial cells from neural stem cells, and using the culture vessel, the neural stem cells are cultured in suspension in a medium containing a growth factor, and cell aggregates are obtained. And a step of adding a differentiation-inducing factor to the medium to adhere the cell aggregate to the culture vessel to differentiate the neural stem cells, the culture vessel having a general formula

(In the formula, R ¹ , R ² and R ³ are each independently an alkyl group having 1 to 6 carbon atoms, and m is an integer of 2 to 6)
Is present near the surface.

According to one embodiment of the present invention, it is possible to provide a culture method capable of forming nerve cells and / or glial cells from a neural stem cell through a cell aggregate using a single culture vessel. .

It is a figure which shows an example of the culture method which concerns on one Embodiment of this invention. 2 is an optical micrograph showing suspension culture of neural stem cells of Example 1. 2 is an optical micrograph showing the differentiation of neural stem cells constituting the cell aggregate of Example 1. FIG.

Next, an embodiment for carrying out the present invention will be described with reference to the drawings.

FIG. 1 shows an example of a culture method according to an embodiment of the present invention.

In this culturing method, a culture vessel D in which the group represented by the general formula (1) is present near the bottom surface is used.

First, a culture medium D containing a neural stem cell C and a growth factor (not shown) is introduced into the culture vessel D (see FIG. 1 (a)). When the neural stem cell C is cultured in suspension, the neural stem cell C grows and aggregates. As a result, a cell aggregate C ′ is formed (see FIG. 1B). This is because the group represented by the general formula (1) existing in the vicinity of the bottom surface of the culture vessel D suppresses the adhesion of the cell aggregate C ′ in addition to the neural stem cell C being a non-adherent cell. It is thought that.

Next, when a differentiation-inducing factor (not shown) is added to the medium M, the cell aggregate C ′ adheres to the bottom surface of the culture vessel D (see FIG. 1C), and the neural stem cells constituting the cell aggregate C ′ C differentiates into neurons and glial cells.

Neural stem cells C are not particularly limited, but neural stem cells derived from mammals such as humans, rats, mice, primates and the like can be used.

The medium is not particularly limited as long as neural stem cells C can be cultured, and examples thereof include a serum-free medium containing an appropriate amount of antibiotics.

The growth factor is not particularly limited as long as the neural stem cell C can be grown, and examples thereof include epithelial cell growth factor and fibroblast growth factor.

The differentiation inducing factor is not particularly limited as long as it can induce differentiation of neural stem cells C constituting the cell aggregate C ′, and examples thereof include fetal bovine serum.

The form of the culture vessel D is not particularly limited, and examples thereof include a dish, a multiwell plate, a flask, a roller bottle, and a device unit having a cell culture process.

In the culture vessel D, a group capable of producing a silanol group by hydrolysis, a silanol group, a hydroxyl group derived from a semi-metal oxide and / or a hydroxyl group derived from a metal oxide is added to a vessel in the vicinity of the surface. It can be produced by reacting a group capable of generating a silanol group by decomposition with a surface modifier having a group represented by the general formula (1). At this time, the surface modifier has a molecular weight of 315 to 650.

As a method of introducing a silanol group by hydrolysis and / or a silanol group in the vicinity of the surface of the container, a polymer having a group capable of generating a silanol group by hydrolysis (hereinafter referred to as hydrolyzate). And a method of applying a coating solution containing alkoxysilane to a container.

When a coating solution containing a hydrolyzable polymer and alkoxysilane is applied to a container, the hydrolyzable polymer and alkoxysilane are hydrolyzed to produce silanol groups. Furthermore, the hydrolyzable polymer is crosslinked by dehydration condensation between silanol groups, and a crosslinked polymer layer into which the silanol groups are introduced is formed. Specifically, after applying the coating liquid to the material, water, acid or alkali is applied or heated. Moreover, after applying water, an acid, or an alkali to a material, you may apply | coat a coating liquid. Furthermore, you may mix water, an acid, or an alkali with a coating liquid. In this case, since hydrolysis occurs in the coating solution, it is preferable to appropriately prepare the coating solution at the time of coating. In addition, when using water, an acid, or an alkali, although you may heat, reaction progresses normally normally at room temperature. Moreover, even if water, an acid or an alkali is not used, the reaction proceeds slowly due to moisture in the atmosphere.

The acid or alkali used for the hydrolysis is not particularly limited as long as it can be hydrolyzed, and two or more types can be mixed and used as an aqueous solution.

As the coating solution, a solution obtained by dissolving or dispersing a hydrolyzable polymer and alkoxysilane in an organic solvent can be used. Examples of the organic solvent include, but are not limited to, aliphatic hydrocarbons, aromatic hydrocarbons, chlorinated hydrocarbons, ethers, alcohols such as 1 to 4 carbon atoms having 1 to 4 carbon atoms, ethyl cellosolve, and butyl cellosolve. Such as cellosolve, dioxane, methyl acetate, diformamide, etc., may be used in combination.

The content of the hydrolyzable polymer in the coating solution is usually 0.001 to 20% by mass, preferably 0.1 to 5% by mass. If the content of the hydrolyzable polymer in the coating solution is less than 0.001% by mass, a sufficient effect may not be obtained by one coating, and if it exceeds 20% by mass, the coating property is lowered. Sometimes.

In addition, the mass ratio of the hydrolyzable polymer to the alkoxysilane is usually 0.01 to 20%, preferably 0.2 to 5%. If the mass ratio of the hydrolyzable polymer to the alkoxysilane is less than 0.01%, the strength of the crosslinked polymer layer may be insufficient. If it exceeds 20%, the silanol groups introduced into the crosslinked polymer layer The amount may be insufficient.

The method for applying the coating solution is not particularly limited, and examples thereof include a dip coating method, a spray coating method, and a spin casting method.

Although it does not specifically limit as a material which comprises a container, Organic materials, such as PP (polypropylene), a cycloolefin resin, a polycarbonate, PET (polyethylene terephthalate), PEEK, a fluorine resin, a polystyrene, a polyvinyl chloride; Gold, titanium, Examples include inorganic materials such as aluminum, iron, copper, stainless steel, alumina, titanium oxide, and zinc oxide.

The hydrolyzable polymer is not particularly limited as long as it is a polymer having a group capable of generating a silanol group by hydrolysis.

(Wherein R ¹ is a hydrogen atom or a methyl group, R ² is an alkylene group having 1 to 6 carbon atoms, preferably a propylene group, and R ³ , R ⁴ and R ⁵ are each independently And an alkoxyl group having 1 to 6 carbon atoms, preferably a methoxyl group or an ethoxyl group.)
A homopolymer or copolymer (hereinafter referred to as polymer (A)) obtained by polymerizing the monomer (A-1) represented by formula (I) can be used. At this time, two or more monomers (A-1) may be used.

Also, when synthesizing the polymer (A), the general formula

(Wherein R ¹ is a hydrogen atom or a methyl group, and R ² is a linear, branched or cyclic alkyl group having 1 to 18 carbon atoms, preferably an alkyl group having 1 to 6 carbon atoms, Particularly preferred is a methyl group.)
The monomer represented by (A-2) may be copolymerized. At this time, two or more monomers (A-2) may be used.

Also, when synthesizing the polymer (A), the general formula

(Wherein R ¹ is a hydrogen atom or a methyl group, R ² is an alkylene group having 1 to 6 carbon atoms, preferably an ethylene group, a propylene group or a 2-hydroxypropylene group, and X is formula

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are each independently a linear or branched alkyl having 1 to 6 carbon atoms. Group, preferably a methyl group.)
A group represented by general formula (X-1):

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a methyl group, R ⁷ is a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a butyl group, and x is a positive integer.)
A group represented by formula (X-2) or a general formula

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are each independently a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a methyl group, R ⁷ is an alkylene group having 1 to 6 carbon atoms, preferably an ethylene group, a propylene group or a 2-hydroxypropylene group, R ⁸ is a hydrogen atom or a methyl group, and y is a positive integer. .)
A group represented by formula (X-3). )
The monomer represented by (A-3) may be copolymerized. When X is a group (X-2) or (X-3), the monomer (A-3) preferably has a molecular weight of 1,000 to 100,000, particularly preferably 2,000 to 20,000. At this time, two or more monomers (A-3) may be used.

Also, when synthesizing the polymer (A), the general formula

(Wherein R ¹ represents a hydrogen atom or a methyl group, R ² represents an alkylene group having 1 to 6 carbon atoms, preferably an ethylene group or a propylene group, and Y represents a general formula

(Wherein R ¹ , R ² and R ³ are each independently an alkyl group having 1 to 6 carbon atoms, preferably a methyl group, and Z ⁻ is a conjugate of a halide ion or an organic or inorganic acid. Ion.)
A group represented by formula (Y-1) or a general formula

(In the formula, R ¹ and R ² are each independently an alkyl group having 1 to 6 carbon atoms, preferably a methyl group.)
A group represented by formula (Y-2). )
The monomer represented by (A-4) may be copolymerized. At this time, two or more monomers (A-4) may be used.

That is, when the polymer (A) is synthesized, the monomer (A-2), the monomer (A-3) and / or the monomer (A-4) may be copolymerized together with the monomer (A-1).

The content of the monomer (A-1) in all monomers used for synthesizing the polymer (A) is preferably 30 to 85% by mass. If the content of the monomer (A-1) in all the monomers is less than 30% by mass, the crosslinking density is lowered and the effect of hydrophilicity may not be sufficiently maintained. The uniformity of the polymer layer may be reduced.

In addition, the content of the monomer (A-2) in all the monomers used for synthesizing the polymer (A) is usually 1 to 75% by mass, and preferably 10 to 60% by mass. If the content of the monomer (A-2) in all monomers is less than 1% by mass, the water resistance of the crosslinked polymer layer may be lowered. If the content exceeds 75% by mass, the polymer (A) is difficult to alcohol. May become soluble.

Furthermore, the content of the monomer (A-3) in all monomers used for synthesizing the polymer (A) is usually 1 to 70% by mass, and preferably 5 to 60% by mass. When the content of the monomer (A-3) in all monomers is less than 1% by mass, the water resistance of the crosslinked polymer layer may be lowered. When the content exceeds 70% by mass, the polymer (A) is difficult to alcohol. May become soluble.

The ratio of the mass of the monomer (A-4) to the total mass of the monomer (A-1), the monomer (A-2) and the monomer (A-3) is usually from 0.01 to 1, 05 to 0.5 are preferred. When this ratio is less than 0.01, the flexibility of the crosslinked polymer layer may be lowered, and when it exceeds 1, the water resistance of the crosslinked polymer layer may be lowered.

The number average molecular weight of the polymer (A) is preferably 2000 to 150,000. When the number average molecular weight of the polymer (A) is less than 2000, the time for forming the crosslinked polymer layer may be long. When the number average molecular weight exceeds 150,000, the viscosity of the coating liquid becomes high and the coating property and workability are poor. Sometimes.

Incidentally, specific examples and production methods of the polymer (A) are disclosed in JP-A-11-302129.

Also, as hydrolyzable polymer, general formula

(Wherein R ¹ represents an alkyl group having 1 to 22 carbon atoms or a phenyl group, preferably a methyl group.)
A homopolymer or copolymer having a structural unit (B-1) represented by the formula (hereinafter referred to as polymer (B)) can be used. At this time, the polymer (B) may have two or more structural units (B-1).

Also, the polymer (B) is a general formula

(Wherein, R ¹ and R ² are each independently an alkyl group having 1 to 22 carbon atoms or a phenyl group, preferably a methyl group.)
The structural unit (B-2) represented by At this time, the polymer (B) may have two or more structural units (B-2).

The polymer (B) preferably has a content of the structural unit (B-1) of 1 to 90% by mass. When the content of the structural unit (B-1) is less than 1% by mass, the crosslinking density is lowered and the effect of hydrophilization may not be sufficiently maintained. When the content exceeds 90% by mass, Uniformity may be reduced.

Furthermore, the polymer (B) preferably has a constituent unit (B-2) content of 10 to 99% by mass. When the content of the structural unit (B-2) is less than 10% by mass, the uniformity of the crosslinked polymer layer may be lowered. When the content is more than 99% by mass, the crosslinking density is lowered, and the effect of hydrophilization. May not last long enough.

The number average molecular weight of the polymer (B) is preferably 2,000 to 500,000. When the number average molecular weight is less than 2,000, the time for forming the crosslinked polymer layer may be long. When the number average molecular weight exceeds 500,000, the viscosity of the coating solution becomes high and the coating property and workability may be inferior.

As the hydrolyzable polymer, the polymer (A) and the polymer (B) may be used in combination, or a hydrolyzable polymer and a non-hydrolyzable polymer may be used in combination. Although it does not specifically limit as a non-hydrolyzable polymer, The polymer (A), polymer (B), etc. which do not have a group which can produce | generate a silanol group by hydrolysis are mentioned.

The method for introducing silanol groups in the vicinity of the surface of the container is not particularly limited, and examples thereof include a method of forming a film containing a silicone resin having a silanol group by applying a coating solution containing a silicone resin to the container. .

The film containing a silicone resin having a silanol group preferably has a water contact angle of 3 to 50 °. It is difficult to form a film having a water contact angle of less than 3 °. When a film having a water contact angle of more than 50 ° is formed, the group represented by the general formula (1) can be introduced at high density. It may disappear.

The silicone resin contained in the coating solution is not particularly limited, but the general formula (R ¹ O) _n Si (R ² ) _4-n
Wherein R ¹ and R ² are each independently an alkyl group having 1 to 8 carbon atoms, n is an integer of 1 to 4, and when n is 1 or 2, a plurality of R ² may be the same or different, and when n is 2 or 3, a plurality of R ¹ may be the same or different.
The resin obtained by condensing after hydrolyzing the alkoxysilane represented by these is mentioned, You may use 2 or more types together. At this time, the silicone resin having a silanol group contained in the film having a water contact angle of 3 to 50 ° may be the same as or different from the silicone resin contained in the coating solution.

The organic solvent contained in the coating solution is not particularly limited, but alcohols such as aliphatic hydrocarbons, aromatic hydrocarbons, chlorinated hydrocarbons, ethers, and 1 to 4 valent aliphatic alcohols having 1 to 4 carbon atoms. , Ethyl cellosolve, cellosolve such as butyl cellosolve, dioxane, methyl acetate, diformamide, etc., may be used in combination.

The content of the silicone resin in the coating solution is usually 0.001 to 1% by mass, preferably 0.1 to 1% by mass. When the content of the silicone resin in the coating solution is less than 0.001% by mass, a uniform film may not be formed, and when it exceeds 20% by mass, applicability may be lowered.

Although it does not specifically limit as a material which comprises a container, Organic materials, such as a polycarbonate, PET (polyethylene terephthalate), a polystyrene, an acrylic resin; Gold, titanium, aluminum, iron, copper, stainless steel, an alumina, a titanium oxide, a zinc oxide, etc. Inorganic materials and the like.

Further, a glass container, a quartz container, or the like may be used as a container in which silanol groups are present near the surface.

The metal oxide constituting the container in which the hydroxyl group derived from the metal oxide is present in the vicinity of the surface is not particularly limited, and examples thereof include titanium oxide, zinc oxide, iron oxide, chromium oxide, and aluminum oxide.

The metal oxide constituting the container in which the hydroxyl group derived from the semimetal oxide is present in the vicinity of the surface is not particularly limited, and examples thereof include germanium oxide, arsenic oxide, and boron oxide.

Examples of the group capable of generating a silanol group by hydrolysis, a silanol group, a hydroxyl group derived from a semi-metal oxide and / or a group reactive to a hydroxyl group derived from a metal oxide include silanol by hydrolysis. Examples include groups capable of generating a group.

In the surface modifier, it is preferable that a group capable of generating a silanol group by hydrolysis is bonded to a group represented by the general formula (1) via a spacer. The spacer is not particularly limited, and examples thereof include an alkylene group having at least one methylene group, oxyethylene group, and amino group.

Examples of the group capable of generating a silanol group by hydrolysis include a hydrosilyl group, an alkoxysilyl group, a halosilyl group, an acyloxysilyl group, and an aminosilyl group. From the viewpoint of stability, reactivity, etc., the number of carbon atoms. Is preferably an alkoxysilyl group or hydrosilyl group of 1 to 6.

The surface modifier is not particularly limited as long as it has a group capable of generating a silanol group by hydrolysis and a group represented by the general formula (1), but JP-A-2006-11380 The compound currently disclosed by the gazette is mentioned.

Next, in a container having a group capable of generating a silanol group by hydrolysis, a silanol group, a hydroxyl group derived from a semi-metal oxide and / or a hydroxyl group derived from a metal oxide in the vicinity of the surface, a general formula ( A method for introducing the group represented by 1) will be described.

First, a surface modifier in a container in which a group capable of generating a silanol group by hydrolysis, a silanol group, a hydroxyl group derived from a semi-metal oxide and / or a hydroxyl group derived from a metal oxide exists in the vicinity of the surface A coating solution containing is applied. At this time, a group capable of generating a silanol group by hydrolysis is hydrolyzed to generate a silanol group. In addition, silanol groups present near the surface of the container, hydroxyl groups generated by hydrolysis of metalloid oxides and / or hydroxyl groups generated by hydrolysis of metal oxides, and generated by hydrolysis of surface modifiers. The surface of the container is modified by dehydration condensation of silanol groups. Specifically, after applying the coating solution to the container, water, acid or alkali is applied or heated. Moreover, after applying water, an acid, or an alkali to a container, you may apply | coat a coating liquid. Furthermore, you may mix water, an acid, or an alkali with a coating liquid. In this case, since hydrolysis occurs in the coating solution, it is preferable to appropriately prepare the coating solution at the time of coating. In addition, when using water, an acid, or an alkali, although you may heat, reaction progresses normally normally at room temperature. Moreover, even if water, an acid or an alkali is not used, the reaction proceeds slowly due to moisture in the atmosphere.

The acid or alkali is not particularly limited as long as a group capable of generating a silanol group by hydrolysis can be hydrolyzed, and two or more kinds may be used in combination. The acid or alkali used for hydrolysis may be used as an aqueous solution.

As the coating solution, a solution obtained by dissolving or dispersing a surface modifying agent in an organic solvent can be used. Examples of the organic solvent include aliphatic hydrocarbons, aromatic hydrocarbons, chlorinated hydrocarbons, ethers, alcohols such as monohydric to tetrahydric aliphatic alcohols having 1 to 4 carbon atoms, cellosolves such as ethyl cellosolve and butyl cellosolve, and dioxane. , Methyl acetate, diformamide and the like.

The content of the surface modifier in the coating solution is usually 0.1 to 30% by mass, preferably 1 to 10% by mass. If the content of the surface modifier in the coating solution is less than 0.1% by mass, the first surface modifier may not be sufficiently applied in one application, and if it exceeds 30% by mass, The applicability may decrease.

In a container in which a group capable of generating a silanol group by hydrolysis, a silanol group, a hydroxyl group derived from a semi-metal oxide and / or a hydroxyl group derived from a metal oxide is present in the vicinity of the surface, the general formula (1 The method disclosed in JP 2011-236345 A may be used as a method for introducing a group represented by

In the culture vessel D, a surface modifying agent having a group represented by the general formula (1) is reacted with an amino group (or carboxyl group) in a vessel having a carboxyl group (or amino group) in the vicinity of the surface. You may manufacture by. At this time, the surface modifier has a molecular weight of 225 to 650.

The method for introducing an amino group in the vicinity of the surface of the container is not particularly limited, and examples thereof include nitrogen plasma treatment, ammonia plasma treatment, a method of reacting a surface treatment agent, and a silicon gas phase treatment.

In the nitrogen plasma treatment, an amino group is introduced in the vicinity of the surface of the container by generating a low temperature plasma in a nitrogen gas atmosphere (for example, Surface and Coatings Technology 116-119 (1999) 802-807, Colloids and Surfaces A: Physicochem. Eng. Aspects 195 (2001) 81-95, Macromol. Chem. Phys. 200.989-996 (1999)). Specifically, the container is accommodated in a reaction container, and after the inside of the reaction container is evacuated with a vacuum pump, nitrogen gas is introduced and glow discharge is performed.

In the ammonia plasma treatment, amino groups are introduced near the surface of the container by generating a low temperature plasma in an ammonia gas atmosphere. Specifically, the container is accommodated in a reaction container, and after the inside of the reaction container is evacuated with a vacuum pump, ammonia gas is introduced and glow discharge is performed.

The material constituting the container is not particularly limited, and examples thereof include polyvinyl chloride, acrylic resin, polystyrene, polypropylene, polyethylene, polyester, cycloolefin resin, polycarbonate, and glass.

In the method of reacting the surface treatment agent, a group capable of generating a silanol group by hydrolysis of an alkoxysilyl group or the like using a surface treatment agent such as alkoxysilane having an amino group, chlorosilane, or silazane, silanol group, An amino group is introduced in the vicinity of the surface of the container in which the hydroxyl group derived from the metalloid oxide and / or the hydroxyl group derived from the metal oxide exists in the vicinity of the surface. Specifically, first, a water / 2-propanol mixed solution is charged into a container, and 3-aminopropyltrimethoxysilane is added, followed by heating to 100 ° C. and reaction for 6 hours. Next, after cooling to room temperature, it is washed with methanol and dried.

The material constituting the container is not particularly limited, but 3-trimethoxysilylpropyl methacrylate-methyl methacrylate-divinylbenzene copolymer, polyvinyl chloride, acrylic resin, polystyrene, polypropylene, polyethylene, polyester, cycloolefin resin , Polycarbonate, silica, glass, alumina, talc, clay, mica, asbestos, titanium oxide, zinc white, iron oxide and the like.

In silicone vapor phase treatment, hydrosilyl groups are introduced in the vicinity of the surface of the container using 1,3,5,7-tetramethylcyclotetrasiloxane, and then the alkene having an amino group is allowed to react with the vicinity of the surface of the container. An amino group is introduced into (see, for example, Japanese Patent Publication No. 1-54379, Japanese Patent Publication No. 1-54380, Japanese Patent Publication No. 1-54381). Specifically, first, 1,3,5,7-tetramethylcyclotetrasiloxane and a container are placed in a desiccator and deaerated with an aspirator. Next, after making it react at 80 degreeC for 16 hours, a container is taken out and it is made to dry at 120 degreeC. Further, the obtained container is immersed in ethanol, allylamine is added, and then an ethanol solution of chloroplatinic acid is added and stirred at 60 ° C. for 2 hours. After the reaction is completed, it is washed with ethanol and dried under reduced pressure.

The material constituting the container is not particularly limited, but styrene-divinylbenzene copolymer, polyvinyl chloride, acrylic resin, polystyrene, polypropylene, polyethylene, polyester, cycloolefin resin, polycarbonate, mica, talc, kaolin, alumina, Examples include titanium oxide, zinc oxide, and iron oxide.

The alkene having an amino group is not limited to allylamine, and may be an amine having a vinyl group, an amine having an acrylic group, or the like. The amino group may be protected with a butoxycarbonyl group, a benzyloxycarbonyl group, or the like. Furthermore, instead of an alkene having an amino group, an alkene having a group capable of introducing an amino group by reaction with, for example, a diamine such as an epoxy group may be used.

The method for introducing a carboxyl group in the vicinity of the surface of the container is not particularly limited, and examples thereof include a method of reacting a surface treatment agent and a silicone gas phase treatment.

In the method of reacting the surface treatment agent, a group capable of generating a silanol group by hydrolysis of an alkoxysilyl group or the like using a surface treatment agent such as an alkoxysilane having a carboxyl group, chlorosilane, or silazane, a silanol group, A carboxyl group is introduced in the vicinity of the surface of the container in which the hydroxyl group derived from the semimetal oxide and / or the hydroxyl group derived from the metal oxide exists in the vicinity of the surface. Specifically, first, triethoxysilylpropyl succinic anhydride is dissolved in N, N-dimethylformamide, distilled water and 4-dimethylaminopyridine are added, and the mixture is stirred at room temperature for 16 hours to give a silane having a carboxyxyl group. A coupling agent is synthesized. Next, a water / 2-propanol mixed solution is charged into a container, a silane coupling agent having a carboxyl group is added, and then heated to 100 ° C. and reacted for 6 hours. Further, after cooling to room temperature, it is washed with methanol and dried.

The material constituting the container is not particularly limited, but 3-trimethoxysilylpropyl methacrylate-methyl methacrylate-divinylbenzene copolymer, silica, glass, alumina, talc, clay, mica, asbestos, titanium oxide, zinc Examples include flower and iron oxide.

In the silicone gas phase treatment, 1,3,5,7-tetramethylcyclotetrasiloxane is used to introduce a hydrosilyl group in the vicinity of the surface of the container, and then react with an alkene having a carboxyl group to thereby react in the vicinity of the surface of the container. (See, for example, Japanese Patent Publication No. 1-54379, Japanese Patent Publication No. 1-54380, Japanese Patent Publication No. 1-54381). Specifically, first, a container containing 1,3,5,7-tetramethylcyclotetrasiloxane is placed in a desiccator and deaerated with an aspirator. Next, after making it react at 80 degreeC for 16 hours, a container is taken out and it is made to dry at 120 degreeC. Further, the obtained container is immersed in ethanol, allylcarboxylic acid is added, an ethanol solution of chloroplatinic acid is added, and the mixture is stirred at 60 ° C. for 2 hours. After the reaction is completed, it is washed with ethanol and dried under reduced pressure.

The material constituting the container is not particularly limited, and examples thereof include styrene-divinylbenzene copolymer, mica, talc, kaolin, alumina, titanium oxide, zinc oxide, and iron oxide.

The alkene having a carboxyl group is not limited to allylcarboxylic acid, and may be any carboxylic acid having a vinyl group, carboxylic acid having an acrylic group, or the like.

In the surface modifier, the amino group (or carboxyl group) is preferably bonded to the group represented by the general formula (1) via a spacer. The spacer is not particularly limited, and examples thereof include an alkylene group having at least one methylene group, oxyethylene group, and amino group.

Next, the surface modifier having an amino group and a group represented by the general formula (1) will be described.

The surface modifier having an amino group and a group represented by the general formula (1) is not particularly limited, but JP-A-2006-7203, JP-A-2006-7204, and JP-A-2008-174491. The compound etc. which are indicated by the gazette are mentioned.

When the carboxyl group existing in the vicinity of the surface of the container and the amino group of the surface modifier are condensed by a general reaction, an amide bond is formed. Specifically, a container is charged with a solution of N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and carboxyl groups existing in the vicinity of the surface of the container are activated, and then the surface modification is performed. Charge the material into the container.

Next, the surface modifier having a carboxyl group and a group represented by the general formula (1) will be described.

The surface modifier having a carboxyl group and a group represented by the general formula (1) is not particularly limited, and examples thereof include compounds disclosed in JP-A-2006-11381.

When the amino group present in the vicinity of the surface of the container and the carboxyl group of the surface modifier are condensed by a general reaction, an amide bond is formed. Specifically, using a solution of N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, a surface modifier having an active esterified carboxyl group is charged into a container.

[Production of culture vessel]
Chemical formula

A solution prepared by mixing 0.6 g of the compound represented by the formula: 0.4 mL of tetraethyl orthosilicate, 1 mL of 1M hydrochloric acid and 10 mL of methanol was dip-coated on a glass substrate, and then dried at room temperature for 1 hour. Next, after washing with water, drying was performed to obtain a glass substrate having phosphorylcholine groups in the vicinity of the surface.

A frame made of silicone resin was attached on a glass substrate having phosphorylcholine groups in the vicinity of the surface to obtain a culture vessel having a bottom area of 1 cm ² .

[Example 1]
D-MEM / F12 medium (manufactured by Invitrogen), 1% by mass of N-2 additive (manufactured by Invitrogen), epidermal growth factor Human recombinant EGF (manufactured by PeproTech), 20 ng / mL, fibroblast growth factor Human recombinant bFGF (manufactured by PeproTech) at 20 ng / mL, Antibiotic-Antimotic (Penicillin-Streptomycin-Amphotericin B mixture) (manufactured by Invitrogen) and medium 1 by mass.

After introducing 0.5 mL of a medium in which mouse-derived neural stem cells are dispersed so that the cell density is 2 × 10 ⁴ cells / mL into a culture vessel and seeding the neural stem cells, the temperature is 37 ° C., carbon dioxide gas The culture vessel was moved to an incubator maintained at a concentration of 5% and humidity of 100%, and neural stem cells were cultured in suspension. As a result, neural stem cells proliferated, migrated, and a cell aggregate was formed about 60 hours after seeding the neural stem cells (see FIG. 2). 2 (a), (b), (c), and (d) are cases where the elapsed time after seeding the neural stem cells is 0 hours, 24 hours, 48 hours, and 72 hours, respectively.

72 hours after seeding the neural stem cells, fetal bovine serum (manufactured by HyClone) was added to the medium so that the concentration was 10% by mass. As a result, about 6 hours after the addition of fetal bovine serum, cell aggregates began to adhere to the glass substrate on which phosphorylcholine groups were present in the vicinity of the surface. Further, about 12 hours after the addition of fetal bovine serum, the neural stem cells constituting the cell aggregates differentiated into nerve cells or glial cells, and flattened to extend the fibrous processes. Furthermore, about 24 hours after adding fetal bovine serum, a large number of neurons and glial cells formed a complex network (see FIG. 3). 3 (a), (b) and (c) are cases where the elapsed time after adding fetal bovine serum is 0 hours, 24 hours and 48 hours, respectively.

This international application claims priority based on Japanese Patent Application 2011-288860 filed on December 28, 2011, and the entire contents of Japanese Patent Application 2011-288860 are incorporated herein by reference. .

C Neural stem cell C 'cell aggregate D Culture vessel M Medium

Claims

A culture method for forming neural cells and / or glial cells from neural stem cells,
Using a culture vessel to form a cell aggregate by suspension culture of neural stem cells in a medium containing a growth factor;
Adding a differentiation-inducing factor to the medium, and allowing the cell aggregate to adhere to the culture vessel to differentiate the neural stem cells;
The culture vessel has a general formula

(In the formula, R 1 , R 2 and R 3 are each independently an alkyl group having 1 to 6 carbon atoms, and m is an integer of 2 to 6)
The culture | cultivation method characterized by the group represented by these existing in the surface vicinity.
In the culture vessel, a group capable of generating a silanol group by hydrolysis, a silanol group, a hydroxyl group derived from a semi-metal oxide and / or a hydroxyl group derived from a metal oxide is added to a vessel in the vicinity of the surface. It is produced by reacting a compound having a group capable of generating a silanol group by decomposition and a group represented by the general formula (1) and having a molecular weight of 315 or more and 650 or less. The culture method according to claim 1.