JP2016171794A - Carrier and cell culture method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a carrier having cell adhesiveness that can be applied for the large scale culture of anchorage-dependent cells.SOLUTION: A carrier for cell culture has a support, and a polyamino acid comprising 80 mass% or more of an α-amino acid residue having a basic group in a side chain, and a binding density of the polyamino acid to the support is 1 μg/m-10,000 μg/m.SELECTED DRAWING: None

Description

  The present invention relates to a carrier and a cell culture method.

  Unlike differentiated cells, stem cells have the ability to divide and self-renew or to differentiate into daughter cells with different phenotypes and functions. The treatment of proliferating useful cells and transplanting the cells into a patient is a treatment method expected in regenerative medicine and cancer immunotherapy, which are remarkable in recent medical progress. In the fields of production of pharmaceuticals using stem cells, gene therapy, regenerative medicine, immunotherapy, etc., it is required to efficiently culture large amounts of cells, tissues, microorganisms, etc. in an artificial environment. As a cell culture technique capable of mass culture, a microcarrier culture method is known in which cells are attached to the surface of a carrier such as particles and the cells are grown. In the microcarrier culture method, cells, culture solution, and microcarriers that serve as cell adhesion scaffolds are introduced into a culture vessel, and the culture solution is stirred intermittently to attach the cells to the surface of the microcarriers for growth. It is a method to make it. Microcarriers are fine particles used as scaffolds (carriers) for culturing adhesion-dependent cells such as stem cells, and conventionally, microcarriers of various materials have been used (Patent Documents 1 to 4).

  However, it has been difficult to say that a sufficiently satisfactory proliferation rate has been obtained by culturing adhesion-dependent cells using conventional microcarriers. Moreover, sufficient evaluation, such as whether it can culture | cultivate, maintaining the capability of a stem cell, is not performed. In addition, microcarriers coated with a matrix containing an extracellular matrix component effective for cell adhesion are also known, but animal-derived proteins should be used as cell scaffolds when transplanting proliferated cells to the human body. Is not preferred in terms of the risk of developing animal-derived infections. Therefore, a chemically synthesized non-animal derived cell culture carrier is desired.

Japanese Patent No. 4595581 JP 2004-254519 A JP 2004-236553 A Special table 2011-529701 gazette

  The problem to be solved by the present invention is to provide a carrier having cell adhesion that can be applied to mass culture of adhesion-dependent cells. Another problem to be solved by the present invention is to provide a cell culture method capable of growing cells using the carrier and recovering the cultured cells with high yield. .

  The problem to be solved by the present invention has been solved by the following means.

<1> A support and a polyamino acid containing 80% by mass or more of an α-amino acid residue having a basic group in the side chain, and the binding density of the polyamino acid to the support is 1 μg / m ² to 10; A carrier for cell culture, which is 000 μg / m ² .
<2> The carrier according to <1>, wherein the end of the polyamino acid is covalently bonded to the surface of the support.
<3> The carrier according to <1> or <2>, wherein the α-amino acid residue is an α-Lys residue.
<4> The carrier according to any one of <1> to <3>, obtained by polymerizing an amino acid monomer using a nucleophilic functional group on the surface of the support as an initiation terminal.
<5> The carrier according to <4>, wherein the nucleophilic functional group is an amino group, a hydroxyl group, or a thiol group, and the amino acid monomer is an N-carboxyamino acid anhydride or an amino acid carbamate compound.
<6> The carrier according to any one of <1> to <5>, wherein the support is a particle.
<7> The carrier according to <6>, wherein the particles are porous.
<8> The carrier according to <6> or <7>, wherein the particles have a volume average particle diameter of 10 to 1000 μm.
<9> The carrier according to any one of <1> to <8>, wherein the specific gravity is 0.9 to 1.2.
<10> The carrier according to any one of <1> to <9>, which is a microcarrier.
<11> A storage unit is prepared, and an introduction step of introducing an adhesion-dependent cell, a culture solution, and the carrier according to any one of <1> to <10> to the storage unit, and the cell adheres And a growth step of growing the cells adhered to the surface of the carrier while the carrier is suspended.
<12> The cell culture method according to <11>, further comprising an adhesion step of bringing the cells into contact with the carrier and adhering the cells to the surface of the carrier between the introduction step and the growth step. .

  According to the present invention, it was possible to provide a carrier having cell adhesion that can be applied to mass culture of adhesion-dependent cells. In addition, according to the present invention, it was possible to provide a cell culture method capable of proliferating cells using the carrier and recovering the cultured cells with high yield.

It is a photograph which shows the cell growth on the particle | grains of Example 1 of the human mesenchymal stem cell line under stationary culture conditions. It is a graph which shows the cell growth on the particle | grains of Example 1 of the human mesenchymal stem cell line under stationary culture conditions. It is a photograph which shows the cell growth on the particle | grains of Example 2 of a human mesenchymal stem cell line under suspension culture conditions. It is a graph which shows the cell growth on the particle | grains of Example 2 of a human mesenchymal stem cell line under suspension culture conditions. It is a figure which shows the expression of the cell surface marker of the human mesenchymal stem cell line grown on the particle | grains of Example 2 on suspension culture conditions. It is a figure which shows the cell differentiation ability of the human mesenchymal stem cell line grown on the particle | grains of Example 2 on a suspension culture condition. It is a photograph which shows the cell growth on the particle | grains of Example 1 of the primary cultured human mesenchymal stem cell under stationary culture conditions. It is a graph which shows the cell growth on the particle | grains of Example 1 of the primary cultured human mesenchymal stem cell under stationary culture conditions. It is a photograph which shows the cell growth on the particle | grains of Example 2 of the primary cultured human mesenchymal stem cell under stationary culture conditions. It is a photograph which shows the cell growth on the particle | grains of Example 1 of the primary cultured human mesenchymal stem cell under suspension culture conditions. It is a graph which shows the cell growth on the particle | grains of Example 1 of the primary cultured human mesenchymal stem cell under suspension culture conditions.

<Carrier for cell culture>
The carrier for cell culture of the present invention comprises a support and a polyamino acid containing 80% by mass or more of an α-amino acid residue having a basic group in the side chain, and the binding density of the polyamino acid to the support is It characterized in that it is a ^{1μg / m 2 ~10,000μg / m 2} .
As the carrier, a microcarrier is preferable. As used herein, “microcarrier” refers to a base material used in a method for attaching and growing cells on the surface of particles.

(Support)
The shape of the support in the present invention is not particularly limited, and may be any of a particle shape, a plate shape, a fiber shape, a rod shape, a chip shape, and the like, preferably a particle shape. The support may be porous or non-porous, but is preferably porous.
Further, as the material of the support, any of natural substances such as polysaccharides including agarose, dextran, cellulose, etc., inorganic substances such as glass, modified glass and ceramic, synthetic polymers such as polystyrene and polymethyl methacrylate, or The combination is mentioned. Among them, polysaccharides and synthetic polymers are preferable, and synthetic polymers are more preferable because they can ensure the floatability by making them porous and have excellent toughness and can reduce breakage during culture.

  The material used in the present invention is preferably a styrene-based polymer that is widely used in the field of cell culture, and is more preferably a styrene-divinylbenzene copolymer because of excellent mechanical strength.

  As the styrene-divinylbenzene copolymer, those capable of being modified with a functional group having nucleophilicity on the support surface containing the polymer are preferable. Specifically, the structural unit (A) derived from a divinylbenzene monomer: 10 to 30% by mass and the structural unit (B) derived from a styrene monomer: 70 to 90% by mass, and the styrene monomer A copolymer in which 30 to 70% by mass of the structural unit (B) derived from is a structural unit (b-1) derived from a styrene monomer having a 3-membered or 4-membered cyclic ether group is preferred. .

  The structural unit (A) may be derived from a divinylbenzene monomer, but is preferably represented by the following formula (1) from the viewpoint of mechanical strength of the carrier, particularly pressure resistance.

[In Formula (1),
R ¹ and R ² each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ³ represents an organic group having 1 to 10 carbon atoms,
Ring Q represents a benzene ring or a naphthalene ring,
p shows the integer of 0-4. ]

In the formula (1), as the carbon number of the alkyl group represented by R ¹ and R ^2, 1 or 2 are preferred. The alkyl group may be linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group.
R ¹ and R ² are preferably hydrogen atoms.

The number of carbon atoms of the organic group represented by R ³ is preferably 1 to 6, more preferably 1 to 3, and even more preferably 1 or 2.
The organic group is preferably an alkyl group. The alkyl group may be linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group.

The ring Q is preferably a benzene ring from the viewpoint of the mechanical strength of the carrier.
p represents an integer of 0 to 4, and 0 or 1 is preferable. In addition, when p is an integer of 2 to 4, p R ³ may be the same or different.

  As the structural unit represented by the above formula (1), those represented by the formula (2) are particularly preferable.

[In formula (2), each symbol is as defined above, R ³ represents an organic group having 1 to 10 carbon atoms, and p represents an integer of 0 to 4. ]

  Examples of the monomer for deriving the structural unit (A) include divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene and the like. These can be used alone or in combination of two or more. Among these, divinylbenzene is particularly preferable from the viewpoint of the reactivity of the polymerization reaction.

The content of the structural unit (A) is 10 to 30% by mass in the total amount of the polymer, but is preferably 12.5 to 27.5% by mass from the viewpoint of mechanical strength and suppressing brittleness. Mass% is particularly preferred.
In addition, content of each structural unit in the polymer in this specification can be measured by ¹³ C-NMR or the like.

(Structural unit (B))
The structural unit (B) derived from the styrenic monomer includes the structural unit (b-1) derived from the styrenic monomer having a 3-membered ring or a 4-membered cyclic ether group in the total amount of the structural unit (B). 30-70 mass% is contained. In the present specification, the “styrene monomer” refers to a styrene monomer other than the divinylbenzene monomer.
As the styrenic monomer having a 3-membered or 4-membered cyclic ether group for deriving the structural unit (b-1), those having a cyclic ether group represented by the following formula (3) are preferable. What is represented by (4) is more preferable.

[In the formula (3), R ⁴ represents a substituted or unsubstituted alkanediyl group having 1 or 2 carbon atoms. ]

[In Formula (4),
R ⁵ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ⁶ represents an alkanediyl group having 2 or 3 carbon atoms,
R ⁷ represents an alkyl group having 1 to 10 carbon atoms,
m and n each independently represent an integer of 0 to 6;
q represents an integer of 0 to 4,
R ⁴ is as defined above and represents a substituted or unsubstituted alkanediyl group having 1 or 2 carbon atoms. ]

In formulas (3) and (4), examples of the alkanediyl group represented by R ⁴ include a methane-1,1-diyl group, an ethane-1,1-diyl group, and an ethane-1,2-diyl group. However, from the viewpoint of reactivity, a methane-1,1-diyl group is preferred. Examples of the substituent that the alkanediyl group may have include a methyl group and an ethyl group.

In formula (4), R ⁵ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. The carbon number of the alkyl group represented by R ⁵ is preferably 1 or 2. The alkyl group may be linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group.
Among these, R ⁵ is preferably a hydrogen atom from the viewpoint of the reactivity of the polymerization reaction.

Examples of the alkanediyl group represented by R ⁶ include ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2-diyl group, and propane. Examples include -1,3-diyl group and propane-2,2-diyl group, and ethane-1,2-diyl group is preferable.

Moreover, from the viewpoint of suppressing the hydrophobicity of the carrier, the alkyl group represented by R ⁷ is preferably 1 to 6, more preferably 1 to 3, and further preferably 1 or 2.
The alkyl group may be linear or branched. As an alkyl group, the same thing as the alkyl group shown by said R < ⁵ > is mentioned, for example.

m and n each independently represent an integer of 0 to 6, and m is preferably an integer of 0 to 3. Moreover, as n, the integer of 0-3 is preferable and 0 or 1 is more preferable. When n is an integer of 2 to 6, n R ^{6 s} may be the same or different.
Moreover, although q shows the integer of 0-4, 0 or 1 is preferable from a viewpoint of suppressing the hydrophobicity of a support | carrier. In addition, when q is an integer of 2-4, q R < ⁷ > may be the same or different.

  Examples of the styrenic monomer having a 3-membered or 4-membered cyclic ether group include vinyl benzyl glycidyl ether, isopropenyl benzyl glycidyl ether, vinyl phenyl butyl glycidyl ether, vinyl benzyloxyethyl glycidyl ether, and the like. These can be used alone or in combination of two or more. Among these, vinyl benzyl glycidyl ether is preferable.

  The content of the structural unit (b-1) is 30 to 70% by mass in the total mass of the structural unit (B) derived from the styrene monomer, but is preferably 35 to 65% by mass, and 40 to 60% by mass. Is more preferable. It is preferable for it to be within the above-mentioned numerical range since a sufficient amount of functional groups for the reaction described later can be introduced into the support.

  Moreover, as a polymer contained in the support body used by this invention, in addition to the said structural unit (b-1) as a structural unit derived from a styrene-type monomer, the structural unit (b) shown by following formula (5) -2) is further preferable.

[In Formula (5),
R ⁸ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
R ⁹ represents an organic group having 1 to 10 carbon atoms,
t shows the integer of 0-5. ]

In formula (5), the carbon number of the alkyl group represented by R ⁸ is preferably 1 or 2. The alkyl group may be linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and a tert-butyl group.
R ⁸ is preferably a hydrogen atom.

The number of carbon atoms of the organic group represented by R ⁹ is preferably 1 to 6, more preferably 1 to 3, and even more preferably 1 or 2 from the viewpoint of suppressing the hydrophobicity of the support.
The organic group is preferably an alkyl group. The alkyl group may be linear or branched. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and a tert-butyl group.

Moreover, although t shows the integer of 0-5, 0-3 are preferable and 0 or 1 is more preferable from a viewpoint of suppressing the hydrophobicity of a support | carrier. Incidentally, when t is integer of 2 to 5, t number of R ⁹ may be different even in the same.

  Examples of the styrenic monomer for deriving the structural unit (b-2) include styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, 4-ethylstyrene, and 4-isopropylstyrene. , 4-tert-butylstyrene and the like. These can be used alone or in combination of two or more.

As content of a structural unit (b-2), 30-70 mass% is preferable in the total mass of the structural unit (B) derived from a styrene-type monomer from a viewpoint of maintenance of particle shape, and 35-65 mass%. Is more preferable, and 40 to 60% by mass is particularly preferable.
As the ratio of the content of the structural unit (b-1) and the structural unit (b-2) [(b-1) :( b-2)], the introduction amount of the reactive functional group and the retention of the particle shape From the viewpoint, the mass ratio is preferably 30:70 to 70:30, more preferably 35:65 to 65:35, and particularly preferably 40:60 to 60:40.

  The content of the structural unit (B) is 70 to 90% by mass in the total amount of the polymer, but 72.5 to 87.5% by mass is preferable from the viewpoint of mechanical strength and brittleness, and 75 to 85 Mass% is particularly preferred. Within the above numerical range, brittleness can be suppressed and mechanical strength is improved.

  The total content of the structural units (A) and (B) is preferably 80 to 100% by mass, more preferably 90 to 100% by mass, and still more preferably 95 to 100% by mass in the polymer contained in the carrier. .

  The support of the present invention may have a group containing a hydroxy group on the surface. For example, it can be obtained by opening a 3-membered or 4-membered cyclic ether group of the structural unit (b-1) remaining after the polyamino acid is fixed. By using such a support, dispersibility in an aqueous medium is improved by hydrophilizing the support, and nonspecific adsorption of the immobilized polyamino acid to the support surface is suppressed. The group containing a hydroxy group is preferably a group in which the 3- or 4-membered cyclic ether group of the structural unit (b-1) is opened, and a 1,2-dihydroxyethyl group is particularly preferable.

  The support is preferably porous particles in order to control the specific gravity and promote the circulation of the culture solution. The volume average pore diameter of the porous particles is preferably from 1 to 1000 nm, more preferably from 10 to 800 nm, and even more preferably from 100 to 500 nm, from the viewpoint of the functional group introduction efficiency due to the increase in the surface area of the particles.

The specific surface area of the support used in the present invention, preferably 0.05~500m ² / g, more preferably 0.1~250m ² / g, more preferably 10~200m ² / g, 50~150m ² / g is more preferable, and 70 to 100 m ² / g is particularly preferable.

  In addition, when the support used in the present invention is a particle, the volume average particle diameter is preferably 10 to 1000 μm, preferably 50 to 500 μm in a wet state in a medium (culture solution) from the viewpoint of cell growth efficiency. More preferably, 100-300 micrometers is especially preferable. Further, the coefficient of variation of the volume average particle diameter is preferably 40% or less, more preferably 30% or less.

  The volume average pore diameter, specific surface area, and volume average particle diameter are values measured by the methods described in Examples described later. The variation coefficient may be measured by, for example, a flow type particle image analyzer.

The support used in the present invention can be obtained, for example, by using each monomer for deriving the above polymer and subjecting it to suspension polymerization in an aqueous medium according to a conventional method.
The suspension polymerization is preferably performed using a radical polymerization initiator. Examples of the radical polymerization initiator include azo series such as azobisisobutyronitrile, azobisisobutyric acid methyl, azobis-2,4-dimethylvaleronitrile, and the like. Initiators are mentioned. The usage-amount of a polymerization initiator is about 0.01-10 mass parts normally with respect to 100 mass parts of monomer total amounts.

  Further, from the viewpoint of obtaining a support as porous particles, suspension polymerization is preferably performed in the presence of a porous agent. Examples of the porosifying agent include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and undecane; alicyclic hydrocarbons such as cyclohexane and cyclopentane; benzene, toluene, xylene, naphthalene, ethylbenzene, and the like Aromatic hydrocarbons; halogenated hydrocarbons such as carbon tetrachloride and tetrachloroethane; butanol, pentanol, hexanol, heptanol, hexanol, 4-methyl-2-pentanol, 2-ethyl-1-hexanol, etc. Aliphatic alcohols; cycloaliphatic alcohols such as cyclohexanol; aromatic alcohols such as 2-phenylethyl alcohol and benzyl alcohol; diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, 2-octanone, cyclohexanone, etc. Ton class; dibutyl ether, diisobutyl ether, anisole, ethers such as ethoxy benzene; isopentyl acetate, butyl acetate, 3-methoxybutyl, esters such as diethyl malonate. The amount of the porous agent used is usually about 70 to 350 parts by mass with respect to 100 parts by mass of the total amount of monomers.

  The suspension polymerization is preferably performed in an aqueous medium. The aqueous medium is a dispersion stabilizer such as a water-soluble polymer or an inorganic salt; a surfactant such as an anionic surfactant, a nonionic surfactant or an amphoteric surfactant; an inorganic polymerization inhibitor or an organic polymerization. A polymerization inhibitor such as an inhibitor may be added. The amount of the aqueous medium used is preferably 600 to 6000 parts by mass with respect to 100 parts by mass of the total amount of monomers.

  The temperature of the polymerization reaction is preferably within 20 ° C. above and below, more preferably within 15 ° C. above and below, centering on the 10-hour half-life temperature + 20 ° C. The polymerization time is usually 5 minutes to 48 hours, preferably 10 minutes to 24 hours.

(Polyamino acid containing 80% by mass or more of an α-amino acid residue having a basic group in the side chain)
Next, a polyamino acid containing 80% by mass or more of an α-amino acid residue having a basic group in the side chain will be described. In the present specification, an α-amino acid refers to an amino acid in which an amino group is also bonded to a carbon (α carbon) to which a carboxy group is bonded. The configuration of the α-amino acid may be L or D.

  The basic group of the α-amino acid residue having a basic group in the side chain is at least one selected from the group consisting of an amino group, a guanidino group, an imidazolyl group, an indolyl group, and a quaternary ammonium group. Preferably, an amino group is more preferable from the viewpoint of pKa.

The carrier of the present invention preferably has a polyamino acid terminal covalently bonded to the surface of the support, and more preferably has a polyamino acid polymer brush formed on the surface of the support. The bond is preferably a non-multipoint bond.
By adopting such a configuration, when the cells are peeled off, the polymer that modifies the surface of the particles is not dropped, and the adhesiveness of the cells can be maintained.

Such a carrier can be obtained, for example, by polymerizing an amino acid monomer using a nucleophilic functional group on the surface of the support as an initiation terminal. Further, it may be chemically bonded to the surface of the support after polymerization of the polyamino acid.
Examples of the nucleophilic functional group include an amino group, a hydroxyl group, and a thiol group, and among these, an amino group is preferable. For the introduction of the nucleophilic functional group, a monomer having a nucleophilic functional group may be used as a monomer constituting the support, but it may be introduced after the support is produced. For example, a nucleophilic functional group may be introduced by reacting a 3-membered or 4-membered cyclic ether group of the obtained support with a monofunctional or bifunctional or more nucleophilic reagent.

  Examples of the nucleophilic reagent include ammonia water, ethylenediamine, 1,2-bis (2-aminoethoxy) ethane, 4-bis (3-aminopropyl) piperazine, o-xylenediamine, m-xylenediamine, and p-xylenediamine. Bis (2-aminoethoxy) diethylsilane, bis (3-aminopropyl) amine, bis (2-aminoethyl) amine, 1,3-bis (aminomethyl) cyclohexane, 1,4-bis (aminomethyl) cyclohexane , Ethylene glycol bis (3-aminopropyl) ether, bis (4-aminophenyl) sulfide, bis (aminomethyl) norbornane, 1,4-bis (4-aminophenoxy) benzene, trimethylenebis (4-aminobenzoate) ), 4,4′-methylenebis (cyclohexylamine), cysta Min, Tris (2-aminoethyl) amine, DL-dithiothreol, dithioerythritol, 1,2-ethanedithiol, 1,3-propanedithiol, 1,2-butanedithiol, 2-aminoethanol, 2-mercaptoethanol Etc.

  As a method for introducing a nucleophilic functional group, for example, a support having a 3-membered ring or a 4-membered cyclic ether group in an aqueous buffer such as water, phosphate buffer, borate buffer, carbonate buffer and the nucleophilic group. The method of making it contact with a sex reagent is mentioned. Moreover, in order to accelerate | stimulate said nucleophilic substitution reaction, you may coexist salts, such as sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, and trisodium citrate.

  The reaction temperature of the nucleophilic functional group introduction reaction is not particularly limited, but is preferably 4 to 150 ° C, more preferably 30 to 130 ° C, and particularly preferably 60 to 80 ° C. The reaction time is not particularly limited, but is preferably 0.5 to 48 hours, more preferably 1 to 24 hours, and particularly preferably 2 to 6 hours. The remaining 3-membered or 4-membered cyclic ether group may be opened by acid treatment with, for example, sulfuric acid in order to suppress subsequent side reactions. After the treatment with sulfuric acid, neutralization with a basic compound such as triethylamine is preferred.

Examples of amino acid monomers to be reacted with a nucleophilic functional group as a polymerization initiation point include N-carboxyamino acid anhydrides and amino acid derivatives such as amino acid carbamate compounds. By using 80% by mass or more of the amino acid structure contained in these compounds having a basic group in the side chain part, based on the total amount of N-carboxyamino acid anhydride or amino acid carbamate compound, the desired polyamino acid can be obtained. Can be introduced.
Among N-carboxyamino acid anhydrides and amino acid carbamate compounds, amino acid carbamate compounds are more preferable because the molecular weight of polyamino acids can be easily controlled.
Examples of the N-carboxyamino acid anhydride or amino acid carbamate compound include compounds of the following formula (6) and formula (7). As the compound of the formula (7), a compound of the formula (7-2) is preferable.

[Where,
R ¹⁰ represents a divalent organic group having 1 or more carbon atoms,
R ¹¹ represents a hydrogen atom or an organic group having 1 or more carbon atoms,
R ¹³ represents an organic group having 1 or more carbon atoms,
Y represents an electron-withdrawing group;
k shows the integer of 0-5. ]

  More specifically, as represented by the following reaction formula, a reaction using an N-carboxyamino acid anhydride as represented by formula (6) and an amino acid carbamate as represented by formula (7) It can be achieved by a reaction using a compound.

[Where,
R ¹² represents an organic group having 1 or more carbon atoms,
X ² represents a nucleophilic functional group,
X ³ represents a divalent group derived from a nucleophilic functional group,
r represents the number of amino acid repeating units and is, for example, 2 or more.
Other symbols are as defined above. ]

In the present invention, R ¹⁰ and R ¹¹ in formula (6) and formula (7) contain a basic group or a group protecting the same in the total amount of N-carboxyamino acid anhydride or amino acid carbamate compound. The desired polyamino acid can be introduced by using 80% by mass or more.
As R ¹⁰ , R ¹¹ , R ¹² , for example, amino groups such as aminobutyl, aminopropyl, guanidinopropyl, methylimidazolyl, methylindolyl, basic groups such as guanidino group, imidazolyl group, indolyl group are protected. Those having a group protected by a group are preferred.
As shown here, when the amino acid has a hydroxyl group, a thiol group, or a plurality of carboxy groups or amino groups, it is preferable to protect those other than those involved in the reaction. The protection method is not particularly limited, but in the case of an amino group, a method of substitution with a carbobenzyloxy group (Z group), a t-butoxycarbonyl group, a benzoyl group, an acetyl group, or the like. In the case of a guanidino group, p- A method for substitution with a toluenesulfonyl group or the like, a method for substitution with a benzyloxymethyl group, a tert-butoxymethyl group, a 2,4-dinitrophenyl group or a trityl group in the case of an imidazolyl group, a formyl group in the case of an indolyl group A method of replacing with such as is exemplified. When a polyamino acid is produced by the method of the present invention without amino group protection of an amino acid having a plurality of amino groups, it can be grafted with a polyamino acid having a dendron structure or a hyperbranch structure.

  The amino acid or amino acid derivative used in the present invention is derived from both natural and non-natural amino acids as long as it contains 80% by mass or more of a basic group or a group having a protected group in the side chain. But you can. For example, natural amino acids such as lysine, ornithine, arginine, histidine, and tryptophan and their active hydrogen-substituted amino acids; unnatural amino acids that are D-isomers of the aforementioned natural amino acids, and the like. Among these amino acids, amino acids that give monomers that exhibit good solubility in the solvent used for polymerization are particularly preferred.

  Examples of organic solvents include amide solvents such as dimethylacetamide, dimethylformamide, and N-methylpyrrolidone; sulfoxide solvents such as dimethyl sulfoxide; ketone solvents such as 2-butanone, methyl ethyl ketone, and acetone; esters such as ethyl acetate and butyl acetate. Examples thereof include nitrile solvents such as acetonitrile; halogen solvents such as chloroform and dichloromethane; ether solvents such as tetrahydrofuran and cyclopentane monomethyl ether; alcohol solvents such as methanol, ethanol, and butanol.

  The reaction may be performed in the presence of a dehydrating agent. Examples of the dehydrating agent include synthetic zeolite such as molecular sieve 4A, molecular sieve 3A, or molecular sieve 5A, anhydrous sodium sulfate, anhydrous magnesium sulfate, or anhydrous calcium sulfate. The reaction can usually be carried out under air, but it is desirable to carry out under an inert gas atmosphere such as argon or nitrogen. This reaction can be carried out under normal pressure, reduced pressure, or increased pressure. The reaction temperature is usually selected from the range of −70 ° C. to 120 ° C., preferably −10 to 100 ° C. The reaction time usually requires 0.1 to 100 hours.

R ¹³ of the amino acid carbamate compound represents an organic group having 1 or more carbon atoms, and is preferably a monovalent hydrocarbon group which may have a substituent. The monovalent hydrocarbon group which may have a substituent may be an alkyl group which may have a substituent, a cycloalkyl group which may have a substituent, or a substituent. Examples thereof include an alkenyl group, a cycloalkenyl group which may have a substituent, an aryl group which may have a substituent, and an aralkyl group which may have a substituent.

  Examples of groups that can be substituted for these hydrocarbon groups include: nitro groups; halogen atoms such as chlorine atoms and fluorine atoms; perfluoroalkyl groups (wherein alkyl groups are straight-chain, A branched or cyclic saturated or unsaturated alkyl group, etc.), a perchloroalkyl group (wherein, the alkyl group includes the same as the perfluoroalkyl group); Ester group; acyl group having 2 to 15 carbon atoms such as acetyl group and benzoyl group; cyano group and the like, nitro group, halogen atom, halogenated alkyl group (wherein alkyl group includes perfluoroalkyl group and The same may be mentioned).

  In the above-mentioned “alkyl group which may have a substituent”, examples of the “alkyl group” include an alkyl group having 1 to 20 carbon atoms, and an alkyl group having 1 to 10 carbon atoms is particularly preferable. Examples of the alkyl group include linear and branched alkyl groups. Specifically, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, A tert-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decanyl group and the like can be mentioned.

In the “cycloalkyl group optionally having substituent (s)”, examples of the “cycloalkyl group” include a cycloalkyl group having 3 to 20 carbon atoms, particularly a cycloalkyl group having 3 to 10 carbon atoms. preferable. Specific examples include a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group.
In the above “optionally substituted alkenyl group”, examples of the “alkenyl group” include alkenyl groups having 2 to 18 carbon atoms, and alkenyl groups having 2 to 10 carbon atoms are particularly preferable. Specific examples include a vinyl group, a propenyl group, and 3-butenyl.

In the “cycloalkenyl group optionally having substituent (s)”, examples of the “cycloalkenyl group” include a cycloalkenyl group having 5 to 18 carbon atoms, and a cycloalkenyl group having 5 to 10 carbon atoms is particularly preferable. . Specific examples include cyclohexenyl, cyclooctenyl, cyclododecenyl and the like.
In the above “optionally substituted aryl group”, examples of the “aryl group” include aryl groups having 6 to 14 carbon atoms, and aryl groups having 6 to 10 carbon atoms are particularly preferable. Specific examples include phenyl, tolyl, naphthyl and the like.

  In the above-mentioned “aralkyl group which may have a substituent”, the “aralkyl group” includes an aralkyl group having 7 to 13 carbon atoms, and an aralkyl group having 7 to 9 carbon atoms is particularly preferable. Specific examples include benzyl, phenethyl, naphthylmethyl, naphthylethyl and the like.

  As the hydrocarbon group, an aryl group which may have a substituent is suitably used. Specifically, diphenyl carbonate, bis (4-nitrophenyl) carbonate, bis (2-nitrophenyl) carbonate, bis (2,4-dinitrophenyl) carbonate, bis (2,4,6-trinitrophenyl) carbonate Hydrocarbon groups formed using bis (pentafluorophenyl) carbonate, bis (4-chlorophenyl) carbonate, bis (2,4-dichlorophenyl) carbonate, bis (2,4,6-trichlorophenyl) carbonate, etc. Among them, diphenyl carbonate is particularly preferably used. In addition, the carbonic acid diester mentioned above can be manufactured by a well-known method, and can use a commercially available thing.

  Finally, for deprotection, for example, a support modified with Z-group-protected poly-L-lysine, hydrogen bromide (30% acetic acid solution), and trifluoroacetic acid are added to the container, and 2 hours. It may be deprotected according to the type of protecting group used, such as shaking at 150 rpm.

In addition, after superposing | polymerizing a polyamino acid, you may make it couple | bond with a support body by the following ring-opening addition reaction. The ring-opening addition reaction can be performed, for example, in a basic aqueous solution. Examples of the basic aqueous solution include a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, and a calcium hydroxide aqueous solution, but other basic organic compounds such as amines may also be used.
The reaction temperature of the ring-opening reaction is usually from 4 to 90 ° C, preferably from 10 to 80 ° C, particularly preferably from 20 to 70 ° C.

  The carrier of the present invention is preferably one represented by the formula (8).

[In Formula (8),
R ¹⁴ represents an alkanediyl group having 1 to 10 carbon atoms bonded to a support (for example, particles),
R ¹⁵ has 3 to 30 carbon atoms (preferably 3 to 21 carbon atoms, more preferably one having one or more ether groups, one or more hydroxy groups or one or more amino groups, or a combination thereof). An organic group having 3 to 12 carbon atoms, particularly preferably 3 to 9 carbon atoms,
R ¹⁶ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms,
R ¹⁷ and R ¹⁸ each independently represents an alkanediyl group having 1 to 8 carbon atoms,
X ⁴ is each independently —NH ₂ , —N ⁺ H ₃ Z, —NHR ¹⁹ , —N ⁺ H ₂ R ¹⁹ Z, —NR ²⁰ R ²¹ , or —N ⁺ R ²² R ²³ R ²⁴ . Indicate
r represents the number of repeating units of amino acid, for example, 2 or more,
Y represents NH ₂ or N ⁺ H ₃ Z.
However, R < ¹⁹ > -R < ²⁴ > shows a C1-C3 alkyl group each independently, and Z shows arbitrary anions. ]

Examples of the alkanediyl group represented by R ¹⁴ include methane-1,1-diyl group, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, and propane. -1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,1-diyl group, butane-1,2-diyl group, butane-1,3- Diyl group, butane-1,4-diyl group, pentane-1,1-diyl group, pentane-1,2-diyl group, pentane-1,3-diyl group, pentane-1,4-diyl group, pentane- 1,5-diyl group, hexane-1,1-diyl group, hexane-1,2-diyl group, hexane-1,3-diyl group, hexane-1,4-diyl group, hexane-1,5-diyl Group, hexane-1,6-diyl group, heptane-1,7 Diyl, octane-1,8-diyl group, nonane-1,9-diyl group, decane-1,10-diyl group and the like. R ¹⁷ and R ¹⁸ may be the same as those described above.
Examples of the alkyl group represented by R ¹⁶ include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group.
Examples of the alkyl group represented by R ^{19 to} R ²⁴ include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group.

In the present invention, the polyamino acid is a polyamino acid containing 80% by mass or more of an α-amino acid residue having a basic group in the side chain, and the content of the α-amino acid residue having a basic group in the side chain is Is preferably 90% by mass or more, and more preferably 95% by mass.
Examples of the amino acid that forms an α-amino acid residue having an amino group in the side chain include lysine, ornithine, arginine, histidine, tryptophan, asparagine, and glutamine. Among them, lysine and ornithine are preferable, and lysine is particularly preferable. As described above, the amino acid is preferably used as an amino acid derivative having a protective group introduced.

  The weight average molecular weight of the polyamino acid is preferably in the range of 200 to 1,000,000, more preferably in the range of 500 to 100,000, and still more preferably in the range of 1,000 to 20,000.

(Polyamino acid bond density)
In the present invention, bond density polyamino acid to the substrate is at ^{1μg / m 2 ~10,000μg / m 2} . The bonding density is preferably ^{50μg / m 2 ~5,000μg / m 2} , 500μg / m 2 ~2,000μg / m 2 is more preferable. In particular, the desired effect of the present invention is improved by setting it to 500 μg / m ² or more.
The polyamino acid bond density can be calculated from the polyamino acid bond amount and the specific surface area of the support.

(Polyamino acid binding amount)
In the present invention, the binding amount of the polyamino acid to the support is preferably 1 mg / g to 1000 mg / g, more preferably 10 mg / g to 750 mg / g, further preferably 30 mg / g to 500 mg / g, 50 mg / g to 300 mg / g is particularly preferred. By setting it as such a range, the desired effect of this invention improves.
What is necessary is just to measure the coupling | bonding amount of a polyamino acid according to the method as described in the Example mentioned later.

  From the viewpoint of using the carrier of the present invention for suspension culture, the specific gravity is preferably close to 1, more preferably 0.9 to 1.2, and particularly preferably about 1.0. When the specific gravity is higher than that of the culture solution (medium), the microcarrier tends to settle, and it is necessary to vigorously stir for suspension culture. On the other hand, if the specific gravity is smaller than that of the culture medium, the tendency of the microcarriers to rise from the culture medium increases, and it is necessary to stir vigorously. Accordingly, the specific gravity of the microcarrier is preferably equivalent to that of the medium (about 1). Thus, in order to adjust the specific gravity of the entire granular support microcarrier, by selecting a porogen during carrier particle polymerization, forming a gel network by crosslinking, and providing an independent air hole inside the particulate support, etc. It is possible to obtain a suitable specific gravity.

  The preferred range of the volume average particle diameter of the support is as described for the support.

<Cell culture method>
In the cell culture method of the present invention, an accommodating part is prepared, an introduction step of introducing the adhesion-dependent cells, the culture solution, and the carrier of the present invention into the accommodating part, and while the carrier to which the cells adhere is suspended. And a growth step of growing the cells adhered to the surface of the carrier. Preferably, an adhesion step is further included between the introduction step and the proliferation step to bring the cells and the carrier into contact with each other and to adhere the cells to the surface of the carrier.
Hereinafter, the cell culture method of the present invention will be described.

  The cells may be suspended and cultured in either serum medium or serum-free medium.

  As the medium, any known medium for cell culture can be used. Specifically, a chemically defined medium such as HamF10 medium, HamF12 medium, Dulbecco's modified MEM (DMEM) medium, RPMI (Roswell Park Memory Institute) -1640 medium, MEM-α medium, CD (Chemically Defined) -CHO medium, etc. , And mixed media thereof. When used as an animal cell culture medium, glutamine 0.5 to 5 mM, antibiotics (penicillin (100 U / mL), streptomycin (100 μg / mL), G418 (0.3 mg / mL)) and the like may be added. .

  The serum-free medium described above may be protein-free in that it does not contain proteins, hydrolysates and components of unknown composition. Serum-free media can include chemically defined media in which all components have a known chemical structure.

  The present invention provides a method for culturing stem cells stably in vitro for long periods of time. Using this method, cells can be expanded continuously between each passage, and the pluripotency of the expanded human stem cell population is maintained for at least more than 5 passages, usually more than 10 passages. .

  In a preferred embodiment, after culturing, at least 60% or more of the stem cells in the culture maintain stem cell properties, such as maintaining stem cell specific marker expression. Alternatively, this may be at least 70% or more, 80% or more, 90% or more, or 95% or more.

(Introduction process)
The cell culture method of the present invention includes an introducing step of preparing a container and introducing the adhesion-dependent cells, the culture medium, and the carrier of the present invention into the container.

  In the present invention, the adhesion-dependent cells used for culture are also referred to as anchorage-dependent cells and substrate-dependent cells, and are a general term for cells that require a scaffold to be attached when the cells proliferate. For example, cells that can be cultured using a microcarrier as a scaffold are not particularly limited. Specific examples of adhesion-dependent cells include mesenchymal stem cells, bone marrow stem cells, stem cells of various tissues, progenitor cells, blast cells, and the like. Moreover, it may be a cell line or a primary cultured cell.

  The container suitable as the container in the present invention is not limited as long as suspension culture described later is possible, but includes a spinner. Spinners are regulated or non-regulated bioreactors, which can be agitated using various agitation mechanisms such as glass ball agitators, impeller agitators, and other suitable agitators. The culture volume of the spinner is generally 20 mL to 500 mL.

(Adhesion process)
The cell culture method of the present invention preferably includes an adhesion step of bringing the cells and the carrier into contact with each other and adhering the cells to the surface of the carrier between the introduction step and the growth step.

The cells may be seeded at any concentration. Typically, the cells are seeded at about 10,000 cells / cm ² to about 500,000 cells / cm ² of a carrier (eg, a microcarrier). For example, the cells may be seeded at about 50,000 cells / cm ² to about 150,000 cells / cm ² of the carrier. However, higher or lower concentrations can be used. Conditions such as incubation time and temperature, CO ₂ and O ₂ levels, growth medium, etc. can be changed as appropriate according to the properties of the cells being cultured.

(Proliferation process)
The cell culture method of the present invention includes a growth step of growing the cells adhered to the surface of the carrier while floating the carrier to which the cells are adhered.

  For example, the cells are detached from the carrier (eg, microcarrier) by an enzyme treatment such as trypsin treatment and transplanted to another carrier (eg, microcarrier). By repeating this process at least once or more, the cells can be continuously expanded to any desired number of generations.

(Differentiation process)
The cell culture method of the present invention can further include a step of inducing differentiation of stem cells obtained after the culture. This can be achieved by placing the carrier-stem cell complex under conditions that induce stem cell differentiation. Alternatively, the cell culture method of the present invention includes a differentiation step of separating stem cells from the carrier after spin culture, and culturing the separated stem cells in a culture without a carrier under conditions that induce differentiation of the stem cells. it can.

  In addition, one or more types of growth factors or other factors may be added to a medium in which cells are incubated with the carrier of the present invention (for example, a microcarrier). These factors can promote cell proliferation, adhesion, self-renewal, differentiation and the like. Examples of factors that may be added to or included in the medium include vascular endothelial growth factor (VEGF), interleukin, nerve growth factor (NGF), erythropoietin, platelet derived growth factor (PDGF), epithelial growth Factor (EGF), activin such as activin A, hematopoietic growth factor, retinoic acid (RA), interferon, fibroblast growth factor such as basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP), peptidic Trans, such as cell growth factor, heparin binding growth factor (HBGF), hepatocyte growth factor (HGF), tumor necrosis factor (TNF), insulin-like growth factor (IGF) I and II, transforming growth factor-β1 (TGFβ1) Examples include forming growth factor and colony stimulating factor.

  The cultured cells may be used for any suitable purpose, including (i) for use in clinical studies, (ii) for therapeutic purposes, (iii) for drug research and toxicity screening, etc. .

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

Each analysis condition in the examples is shown below.
(Volume average particle size)
The volume average particle diameter of the particles is such that the particles are suspended in pure water so that the concentration is 10% by mass, and a laser diffraction scattering type particle size distribution analyzer (LS13320, manufactured by Beckman Coulter, Inc.) is used with this suspension. Measured with

(Volume average pore diameter and specific surface area)
The volume average pore diameter and specific surface area of the particles were measured using a mercury porosimeter (Autopore IV9520 manufactured by Shimadzu Corporation) for the dried particles obtained by vacuum drying the particles for 24 hours at 40 ° C., and the pore diameter measurement range was 10 to 5000 nm. It was measured.

(Polylysine binding amount)
The binding amount of polylysine, which is a ligand bound to the particles, was quantified with a reagent set using a bicinchoninic acid (BCA) reagent. Specifically, 4 mg of particles in terms of solid content was collected in a test tube, and this was quantified with a BCA Protein Assay Kit from Thermo Fisher Scientific. The reaction was performed by inversion mixing at 37 ° C. for 30 minutes. The calibration curve was prepared using α-poly L lysine having a weight average molecular weight of 30,000.

[Example 1]
To 360.59 g of pure water, 2.17 g of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., GH-23) and 0.036 g of sodium dodecylsulfate (Emar 10G manufactured by Kao Co., Ltd.) are added and stirred overnight to obtain an aqueous solution (α ) Was prepared. On the day of polymerization, another 10 g was extracted.
Next, 4.93 g of 4-vinylbenzylglycidyl ether (manufactured by Toray Fine Chemical Co., Ltd.), 4.93 g of styrene (manufactured by Nippon Oxirane Co., Ltd.), and 2.46 g of divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd.) were mixed with diisobutyl ketone (Wako Pure Chemical Industries, Ltd.). Dissolved in 24.74 g) to prepare an organic monomer solution. In addition, when the total mass of each monomer is 100 parts by mass, the number of parts of each monomer is 40 parts by mass of 4-vinylbenzylglycidyl ether, 40 parts by mass of styrene, and 20 parts by mass of divinylbenzene.
Next, almost the entire amount of the aqueous solution (α) was put into a 500 mL separable flask, a thermometer, a stirring blade, and a cooling pipe were attached, set in a hot water bath, and stirring was started at 380 rpm in a nitrogen atmosphere. After 20 minutes, the entire amount of the organic monomer solution was added to the flask and stirred for another 20 minutes. Subsequently, the separable flask was heated with a hot water bath, and when the internal temperature reached 70 ° C., 1.51 g of 2,2′-azodiisobutyronitrile (initiator) was charged into the flask using a dropping funnel. The initiator remaining in the funnel was washed with 5 g of the extracted aqueous solution (α) and stirred for 5 hours. Thereafter, 1.51 g of 2,2′-azodiisobutyronitrile was put into the flask using a dropping funnel and stirred for 2 hours.
Next, after cooling the reaction solution, the reaction solution was filtered with Nutsche and washed with pure water and isopropanol. The washed particles were dispersed in isopropanol and applied to a 180 μm metal mesh to remove large particles, and subsequently applied to a 90 μm metal mesh to remove small particles. The dispersion was filtered with Nutsche and vacuum dried at 40 ° C. for 8 hours to obtain porous particles (1-1) (particle dry mass 8.3 g). The obtained porous particles (1-1) had a volume average particle diameter of 145 μm, a volume average pore diameter of 171 nm, and a specific surface area of 93 m ² / g.

  4.00 g of porous particles (1-1), 29.12 g of 25% aqueous ammonia (manufactured by Wako Pure Chemical Industries, Ltd.), and 8.00 g of pure water were added to a polypropylene container. The polypropylene container was placed in an incubator shaker and shaken at 70 ° C. for 4 hours at 150 rpm. The reaction solution was filtered and washed with pure water. The total amount of the washed particles and 40.00 g of 0.5 mol / L sulfuric acid were added to another polypropylene container, placed in an incubator shaker, and shaken at 70 ° C. for 2 hours at 150 rpm. Subsequently, the dispersion was filtered, and the total amount of particles obtained as a residue, 36 g of pure water, and 4 g of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) were added to another polypropylene container and placed in a rotor for 30 minutes at 40 rpm. The mixture was stirred under the following conditions. The dispersion was filtered, and further washed and filtered with pure water, a 50 v / v% ethanol aqueous solution, and ethanol. The collected particles were vacuum-dried at 40 ° C. for 8 hours to obtain porous particles (1-2) whose surface was aminated.

Under a nitrogen atmosphere, 4.00 g of porous particles (1-2), 3.80 g of Nα-phenoxycarbonyl-Nε-carbobenzoxy-L-lysine, and N, N-dimethylacetamide (manufactured by Wako Pure Chemical Industries) 207. 29 g was put into a 500 mL separable flask, a thermometer, a stirring blade, and a condenser tube were attached, set in an oil bath, and reacted at 100 ° C. for 4 hours at 150 rpm. After cooling, the dispersion was filtered with a Kiriyama funnel and washed with N, N-dimethylacetamide and isopropanol. The collected particles were vacuum-dried at 40 ° C. for 8 hours to obtain particles whose surface was modified with Z-group-protected poly-L-lysine.
Total amount of particles modified with the above Z-group-protected poly-L-lysine, hydrogen bromide (30% acetic acid solution) (Tokyo Kasei Co., Ltd.) 14.21 g, and trifluoroacetic acid (Wako Pure Chemical Industries, Ltd.) 30.24 g The mixture was added to a polypropylene container and shaken with a rotor at 150 rpm for 2 hours. The dispersion was filtered with a Kiriyama funnel, and washed and filtered with pure water and 1M aqueous sodium hydrogen carbonate solution. Thereafter, the total amount of particles obtained as a residue was immersed in a 1M aqueous sodium hydrogen carbonate solution overnight, and then washed and filtered with pure water and isopropanol. The collected particles were vacuum-dried at 40 ° C. for 8 hours to obtain porous particles (1-3) whose surfaces were modified with poly-L-lysine. The amount of introduced poly-L-lysine was 96.0 mg / g-beads.

[Example 2]
To 360.89 g of pure water, 2.17 g of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd., GH-23) and 0.036 g of sodium dodecylsulfate (Emar 10G manufactured by Kao Corporation) are added and stirred overnight to obtain an aqueous solution (β ) Was prepared. On the day of polymerization, another 10 g was extracted.
Next, 4.19 g of 4-vinylbenzylglycidyl ether (manufactured by Toray Fine Chemical Co., Ltd.), 4.19 g of styrene (manufactured by Nippon Oxirane Co., Ltd.), and 2.10 g of divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd.) were added to diisobutyl ketone (Wako Pure Chemical Industries, Ltd.). Dissolved in 26.31 g) to prepare an organic monomer solution. In addition, when the total mass of each monomer is 100 parts by mass, the number of parts of each monomer is 40 parts by mass of 4-vinylbenzylglycidyl ether, 40 parts by mass of styrene, and 20 parts by mass of divinylbenzene.
Next, almost the entire amount of the aqueous solution (β) was put into a 500 mL separable flask, a thermometer, a stirring blade and a cooling pipe were attached, set in a hot water bath, and stirring was started at 280 rpm in a nitrogen atmosphere. After 20 minutes, the entire amount of the organic monomer solution was added to the flask and stirred for another 20 minutes. Subsequently, the separable flask was heated with a hot water bath, and when the internal temperature reached 70 ° C., 2,2′-azodiisobutyronitrile (initiator) (manufactured by Wako Pure Chemical Industries, Ltd.) 1 using a dropping funnel 1 .23 g was put into a flask and stirred for 5 hours. Thereafter, 1.23 g of 2,2′-azodiisobutyronitrile was put into the flask using a dropping funnel and stirred for 2 hours.
Next, after cooling the reaction solution, the reaction solution was filtered with Nutsche and washed with pure water and isopropanol. The washed particles were dispersed in isopropanol and applied to a 125 μm metal mesh to remove small particles. The dispersion was filtered with Nutsche and vacuum dried at 40 ° C. for 8 hours to obtain porous particles (2-1) (particle dry mass 8.8 g). The volume average particle diameter of the obtained porous particles (2-1) was 269 μm, the volume average pore diameter was 445 nm, and the specific surface area was 96 m ² / g.

  4.00 g of porous particles (2-1), 29.12 g of 25% ammonia water (manufactured by Wako Pure Chemical Industries, Ltd.) and 8.00 g of pure water were added to a polypropylene container. The polypropylene container was placed in an incubator shaker and shaken at 70 ° C. for 4 hours at 150 rpm. The reaction solution was filtered and washed with pure water. The total amount of the washed particles and 40.00 g of 0.5 mol / L sulfuric acid were added to another polypropylene container, placed in an incubator shaker, and shaken at 70 ° C. for 2 hours at 150 rpm. Subsequently, the dispersion was filtered, and the total amount of particles obtained as a residue, 36 g of pure water, and 4 g of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) were added to another polypropylene container and placed in a rotor for 30 minutes at 40 rpm. The mixture was stirred under the following conditions. The dispersion was filtered, and further washed and filtered with pure water, a 50 v / v% ethanol aqueous solution, and ethanol. The collected particles were vacuum-dried at 40 ° C. for 8 hours to obtain porous particles (2-2) whose surface was aminated.

  In the same procedure as in Example 1 except that the porous particles (1-2) are changed to porous particles (2-2), particles whose surface is modified with Z-group-protected poly-L-lysine are obtained, Furthermore, porous particles whose surface was modified with poly-L-lysine were obtained. Let this porous particle be a porous particle (2-3). The amount of poly-L-lysine introduced was 133 mg / g-beads.

[Comparative Example 1]
Under a nitrogen atmosphere, n-butylamine 11 mg (0.15 mmol), Nα-phenoxycarbonyl-Nε-carbobenzoxy-L-lysine 3.00 g (7.49 mmol), N, N-dimethylacetamide (manufactured by Wako Pure Chemical Industries, Ltd.) 15 g was put into a 500 mL separable flask, a thermometer, a stirring blade, and a condenser tube were attached, set in an oil bath, and reacted at 100 ° C. for 4 hours at 130 rpm. After cooling, the reaction solution was added to 1 L of diethyl ether to precipitate, and the resulting solid was filtered off with a Kiriyama funnel. The collected solid was vacuum-dried at 40 ° C. for 8 hours to obtain Z-group-protected poly-L-lysine.
The total amount of the above Z-group protected poly-L-lysine, hydrogen bromide (30% acetic acid solution) (manufactured by Tokyo Chemical Industry Co., Ltd.) 2.10 g, and trifluoroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) 4.20 g were put in a glass container. After stirring for 2 hours with a stirrer and cooling, the reaction solution was added to 1 L of diethyl ether to precipitate, and the resulting solid was filtered off with a Kiriyama funnel. The collected solid was vacuum-dried at 40 ° C. for 8 hours to obtain poly-L-lysine hydrobromide (A) (hereinafter also simply referred to as polymer (A)). This reaction mixture was subjected to NMR quantification to confirm that poly-L-lysine hydrobromide was obtained. The result of the average molecular weight measurement by GPC was Mn5,400 and Mw7,400.

  1.00 g of porous particles (1-1), 0.078 g of polymer (A), 0.076 g of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) and 15.00 g of pure water were added to a polypropylene container. The polypropylene container was placed in an incubator shaker and shaken at 60 ° C. for 8 hours at 150 rpm. The reaction solution was filtered through a Kiriyama funnel and washed with pure water and 1M aqueous sodium hydrogen carbonate solution. Thereafter, the particles were immersed in a 1M aqueous sodium hydrogen carbonate solution overnight, and then washed with pure water and isopropanol. The collected particles were vacuum-dried at 40 ° C. for 8 hours to obtain porous particles (1′-A) whose surfaces were modified with α-poly-L-lysine. The amount of α-poly-L-lysine introduced was 23.7 mg / g-beads.

[Comparative Example 2]
A poly-L-lysine hydrobromide (B) (hereinafter also simply referred to as polymer (B)) was obtained in the same manner as in Comparative Example 1 except that the amount of n-butylamine was changed to 22 mg. This reaction mixture was subjected to NMR quantification to confirm that poly-L-lysine hydrobromide was obtained. The result of the average molecular weight measurement by GPC was Mn2,400 and Mw4,400.
Further, a porous particle (1′-B) whose surface was modified with α-poly-L-lysine was carried out in the same manner as in Comparative Example 1 except that the polymer (A) was changed to the polymer (B). Got. The amount of α-poly-L-lysine introduced was 12.3 mg / g-beads.

[Comparative Example 3]
A poly-L-lysine hydrobromide (C) (hereinafter also simply referred to as polymer (C)) was obtained in the same manner as in Comparative Example 1 except that the amount of n-butylamine was changed to 33 mg. This reaction mixture was subjected to NMR quantification to confirm that poly-L-lysine hydrobromide was obtained. The results of the average molecular weight measurement by GPC were Mn1,400 and Mw2,800.
Further, a porous particle (1′-C) whose surface was modified with α-poly-L-lysine was carried out in the same manner as in Comparative Example 1 except that the polymer (A) was changed to the polymer (C). Got. The amount of α-poly-L-lysine introduced was 6.0 mg / g-beads.

The results of analyzing the particles obtained in each Example and Comparative Example are shown in Table 1 below. The density of poly-L-lysine on the particle surface was determined by the following equation.
[Density (mg / m ² )] = [Poly-L-lysine bond amount (mg / g-bead)] / [Specific surface area (m ² / g-bead)]

[Evaluation using cell lines]
<Evaluation 1-1: Cell Proliferation on Particles of Human Mesenchymal Stem Cell Line under Static Culture Conditions>
When the porous particles (1-3) of Example 1 and a human mesenchymal stem cell line were mixed and seeded in a low adhesion dish (SUMILON, 24 well) and cultured, the human mesenchymal stem cells adhered to the particles. Proliferation was started (FIG. 1, Day 1). The cell growth was monitored by sampling a small amount of particles during culture, staining with Hoechst to visualize the cell nuclei on the particles, and then observing with a fluorescence microscope.
Further, after 3 days, the cells visibly proliferated (FIG. 1, Day 3), and on the 5th and 7th days, it was observed that the cells and particles adhered to each other to form a mixed mass (FIG. 1). , Day 5 and 7). The results of counting the number of cells on days 0 to 7 are shown in FIG. From this result, it was confirmed that the carrier of the present invention showed a remarkable growth rate and could be used effectively as a carrier for cell culture.

  Moreover, when the same test as the above was performed using the porous particles (2-3) of Example 2, the porous particles (2-3) were also the same as the porous particles (1-3). It was confirmed that a result was obtained. Similarly, adhesion and proliferation of human mesenchymal cells were observed for the particles of Comparative Examples 1 to 3.

<Evaluation 1-2: Cell proliferation on particles of human mesenchymal stem cell line under suspension culture conditions>
Add 1 mL of porous particles (2-3) of Example 2 and 50 mL of medium to a spinner flask, add a human mesenchymal stem cell line (1 × 10 ⁶ cells) suspended in 50 mL of medium, and allow to stand overnight. The next day, 1 mL of the particles was collected, and the cells adhered to the particles were stained with Hoechst and observed using a fluorescence microscope. Then, spin culture was started at 45 rpm. After 7 days, 14 days, and 21 days after seeding, the particles were washed with PBS, and the cells were detached by trypsin / EDTA treatment. Continued. In addition, 1 mL of particles were collected 1 day, 4 days, 7 days, 8 days, 11 days, 14 days, 15 days, 18 days and 21 days after sowing.
As a result, it was observed that the human mesenchymal stem cell line grew on the particles even under subculture conditions (FIG. 3).
Moreover, the test similar to the above was done using the particle | grains of Example 1 and Comparative Examples 1-3.

As a result of the above test, it was confirmed that when the particles of Examples 1 and 2 were used, the number of cells adhered at the initial stage was remarkably increased by suspension culture for a long time. As an example, FIG. 3 shows the state of cell proliferation on the particles of Example 2 of a human mesenchymal stem cell line under suspension culture conditions.
On the other hand, for the particles of Comparative Examples 1 to 3, even when suspended in culture for a long time, the growth of the cells adhered at the initial stage was not observed due to the dropping of the cells during the culture.
That is, the particles of Examples 1 and 2 in which poly-L-lysine is graft-polymerized from the functional group on the base particle are more cells than the particles of Comparative Examples 1 to 3 in which poly-L-lysine is bonded at multiple points The adhesion of was good. These results are thought to be due to the difference in the mode of binding of polyamino acids on the particle surface. Therefore, when the same amount of poly-L-lysine immobilized both-bond type particles were compared, the graft polymerized particles showed better growth than the multi-point bonded particles. This is presumably because when the poly-L-lysine main chain on the carrier surface extends in a brush shape and is denser, the cells are more easily recognized as a scaffold.

<Evaluation 1-3: Quantification of proliferating cells on particles of human mesenchymal stem cell line under suspension culture conditions>
Particles collected on the 1st, 4th, 7th, 8th, 11th, 14th, 15th, 18th, and 21st days after sowing collected in Evaluation 1-2 The cells were detached by Trypsin / EDTA treatment and the number of cells was counted.
As a result, as shown in the graph of FIG. 4, although some cell loss was observed at the time of cell passage, the cells continuously proliferated on the particles for 3 weeks after the start of culture, and 490 times the initial seeding amount in 3 weeks. It was possible to grow to

<Evaluation 1-4: Expression of cell surface marker of human mesenchymal stem cell line grown under suspension culture conditions>
For cells suspended in culture on the porous particles (2-3) of Example 2 in Evaluation 1-2, the cells were collected by Trypsin / EDTA treatment after 3 weeks of culture, and expression of various human mesenchymal stem cell surface markers was observed. Observation was performed by immunofluorescence staining.
As a result, human mesenchymal stem cells subcultured on particles were also positive for CD44, CD90, and CD105, while negative for hematopoietic stem cell marker CD34 and lymphocyte marker CD45 (FIG. 5). ), It was confirmed that expression of cell surface markers specific to human mesenchymal stem cells was maintained.

<Evaluation 1-5: Cell differentiation ability of human mesenchymal stem cell line grown under suspension culture conditions>
Regarding cells suspended in culture on the porous particles (2-3) of Example 2 in Evaluation 1-2, the cells were collected by Trypsin / EDTA treatment after 3 weeks of culture, and bone that is the differentiation ability of human mesenchymal stem cells Differentiation ability and fat differentiation ability were evaluated. Regarding bone differentiation, the cells were evaluated by alizarin red staining after induction of differentiation in Lonza's bone differentiation-inducing medium for 15 days. Regarding adipose differentiation, differentiation induction was performed for 17 days in Lonza adipose differentiation induction medium, and then cells were evaluated by Oil red O staining.
As a result, human mesenchymal stem cells cultured in suspension on the particles also differentiated into positive osteoblasts by alizarin red staining in the same manner as cells cultured in commercially available plastic dishes for adherent cell culture. Oil red Since it showed the ability to differentiate into adipocytes stained with O (FIG. 6), it was confirmed that the cells can be cultured while maintaining their properties by culturing on the porous particles (2-3). It was.

[Evaluation using primary cultured cells]
<Evaluation 2-1: Cell proliferation on particles of primary cultured human mesenchymal stem cells under static culture conditions>
In a low adhesion dish (SUMILON, 24 well), the porous particles (1-3) of Example 1 and primary cultured human mesenchymal stem cells were mixed and seeded and cultured. The cell growth was monitored by sampling a small amount of particles during culture, staining with Hoechst to visualize the cell nuclei on the particles, and then observing with a fluorescence microscope.
In addition, after 3 days, the cells started to adhere to the particles and proliferated, and the number of cells started to increase. On the 7th and 16th days, the cells and particles adhered to each other to form a mixed mass (Fig. 7). The results of counting the number of cells on days 0 to 16 are shown in FIG. From this result, it was confirmed that the carrier of the present invention showed a remarkable growth rate and could be used effectively as a carrier for cell culture.

  Moreover, when the same test as the above was performed using the porous particles (2-3) of Example 2, the porous particles (2-3) were also the same as the porous particles (1-3). It was confirmed that a result was obtained (FIG. 9). Adhesion and proliferation of primary cultured human mesenchymal stem cells were also observed in the particles of Comparative Examples 1 to 3.

<Evaluation 2-2: Cell proliferation on primary human mesenchymal stem cells under suspension culture conditions and quantification of proliferating cells>
Using primary cultured human mesenchymal stem cells instead of human mesenchymal stem cell lines and porous particles of Example 1 instead of the porous particles of Example 2 on the 10th and 20th days after seeding, respectively. An evaluation test similar to Evaluation 1-2 and Evaluation 1-3 was performed except that the observation and the number of cells were counted.
As a result, primary cultured human mesenchymal stem cells proliferated on the porous particles (2-3) of Example 1 even under suspension culture conditions (FIGS. 10 and 11).

  As a result of the above test, it was confirmed that the number of cells adhering to the initial stage was remarkably increased in primary cultured human mesenchymal stem cells by carrying out suspension culture for a long time using the carrier of the present invention.

Claims (12)

A support and a polyamino acid containing 80% by mass or more of an α-amino acid residue having a basic group in the side chain,
Carrier for cell culture, wherein the binding density of the polyamino acid to the support is ^{1μg / m 2 ~10,000μg / m 2} .   The carrier according to claim 1, wherein the end of the polyamino acid is covalently bonded to the surface of the support.   The carrier according to claim 1 or 2, wherein the α-amino acid residue is an α-Lys residue.   The carrier according to any one of claims 1 to 3, obtained by polymerizing an amino acid monomer using a nucleophilic functional group on the surface of the support as an initiation terminal. The nucleophilic functional group is an amino group, a hydroxyl group or a thiol group;
The carrier according to claim 4, wherein the amino acid monomer is an N-carboxyamino acid anhydride or an amino acid carbamate compound.   The carrier according to any one of claims 1 to 5, wherein the support is a particle.   The carrier according to claim 6, wherein the particles are porous.   The carrier according to claim 6 or 7, wherein the particles have a volume average particle diameter of 10 to 1000 µm.   The carrier according to any one of claims 1 to 8, wherein the specific gravity is 0.9 to 1.2.   The carrier according to any one of claims 1 to 9, which is a microcarrier. An introduction step of preparing a storage part, introducing the adhesion-dependent cells, the culture solution, and the carrier according to any one of claims 1 to 10 into the storage part,
A cell culturing method comprising the step of growing the cells adhered to the surface of the carrier while floating the carrier to which the cells are adhered.   The cell culture method according to claim 11, further comprising an adhesion step of bringing the cells and the carrier into contact with each other and adhering the cells to the surface of the carrier between the introduction step and the growth step.