JP7589650B2 - Cell culture medium additives and their uses - Google Patents

Description

The present invention relates to a cell culture medium additive suitable for increasing the production of a physiologically active substance during animal cell culture. The present invention further relates to a method for producing a physiologically active substance using a medium containing the cell culture medium additive.

In regenerative medicine using stem cells such as iPS cells, ES cells, and mesenchymal stem cells, it is expected that these cells will be efficiently proliferated and then differentiated into the target tissue for transplantation. In addition, it has become increasingly common to mass-produce useful physiologically active substances (hormones, neurotransmitters, cytokines, vitamins and minerals, enzymes, nucleic acids, etc.) such as monoclonal antibodies on an industrial scale by mass-culturing animal cells. In such cases, animal serum such as fetal bovine serum (FBS) is frequently used as a growth factor. Specifically, a culture medium in which about 10-20% animal serum such as fetal bovine serum (FBS) is added to a synthetic basal medium such as RPM11640, Eagle's MEM, or Ham's F12 is generally used.

However, adding serum such as fetal bovine serum (FBS) to culture media has the disadvantages of increasing culture medium costs and making it difficult to isolate the desired physiologically active substances from culture media that contain serum-derived proteins. Furthermore, there is often variation in quality between lots of serum. Therefore, it is necessary to thoroughly check the lot before using serum and select a serum that can be used, but this selection of serum requires a lot of effort.

In order to solve the above problems, medium additives that can improve the efficiency of cell culture and reduce costs have been investigated.
For example, Patent Document 1 discloses that the addition of glycylglycine to a medium can cause animal cells to produce useful substances at high concentrations.
Furthermore, Patent Document 2 discloses that when cells are cultured in a medium containing a polymer having a structure similar to phospholipids, a physiologically active substance can be efficiently produced.
Furthermore, Patent Document 3 discloses that a medium containing a hydrophobic D-amino acid promotes cell growth and increases the production of useful substances.
In addition, as a study focusing on betaine polymers similar to those of the present invention, Patent Document 4 discloses a protein aggregation inhibitor containing a sulfobetaine polymer as an active ingredient. Patent Document 5 discloses a non-adhesive material for biological materials containing a sulfobetaine polymer. And Patent Document 6 discloses a medical material containing a carbobetaine polymer.

Japanese Patent Application Publication No. 7-23780 Japanese Unexamined Patent Publication No. 4-304882 JP 2015-027265 A International Publication No. WO2016/181975 Patent No. 6353923 Patent No. 4961133

However, many of the conventional serum-free media are not sufficient in terms of proliferation, survival, and antibody productivity of antibody-producing cells, compared to serum-containing media. In addition, Patent Documents 4 to 6 disclose betaine polymers as in the present invention, but do not focus on the cell activity and productivity of physiologically active substances per amount of DNA that are addressed in the present invention.

In view of the above circumstances, the object of the present invention is to provide a medium additive for antibody-producing cells, a method for culturing antibody-producing cells using the additive, a cell medium, and a method for producing antibodies, which increase antibody productivity without increasing costs or labor.

That is, the present invention relates to any one of the following inventions.
The present invention relates to a cell culture medium additive comprising a water-soluble vinyl polymer (a) having at least any one of the structural units represented by the following general formulas 1 to 3 in its side chain:



(Wherein,
_R2 is an alkylene group having 1 to 6 carbon atoms;
R ₃ and R ₄ each independently represent an alkyl group having 1 to 4 carbon atoms;
_R5 is an alkylene group having 1 to 4 carbon atoms; X is an oxygen atom or -NH-;
Y is ^-COO- or _-SO3- ^;
_R7 is a hydrogen atom or a methyl group;
R ₈ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms;
four of R ₁₀ to R ₁₄ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, one of R ₁₀ to R ₁₄ represents a bonding position with the main chain of a vinyl polymer;
R ₁₅ represents an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms;
* indicates the bonding position with the main chain of the vinyl polymer.)

The present invention also relates to the cell culture medium additive, in which the water-soluble vinyl polymer (a) contains 30 to 95 mass% of at least one of the structural units represented by general formulas 1 to 3 relative to 100 mass% of the total monomer units constituting the water-soluble vinyl polymer (a).

The present invention also relates to the cell culture medium additive, in which the water-soluble vinyl polymer (a) contains structural units based on a monomer (B) having a partition coefficient LogP of 0.8 to 10, and the structural units based on the monomer (B) account for 5 to 70% by mass relative to 100% by mass of the total monomer units constituting the water-soluble vinyl polymer (a).

The present invention relates to the aforementioned cell culture medium additive, wherein the water-soluble vinyl polymer (a) is the following (a1) or (a2):
(a1) A copolymer of monomers containing at least any one of monomers (A1) to (A3) represented by the following general formulas 4 to 6.
(a2) A reaction product of a copolymer of at least any one of monomers (A4) to (A6) represented by the following general formulas 7 to 9 and one or more betaining agents (D) selected from the group consisting of cyclic sulfonate esters (D1), ω-halogenated alkylsulfonate metal salts (D2), cyclic carboxylate esters (D3) and ω-halogenated alkylcarboxylate metal salts (D4).






(Wherein,
_R1 is a hydrogen atom or a methyl group;
_R2 is an alkylene group having 1 to 6 carbon atoms;
R ₃ and R ₄ each independently represent an alkyl group having 1 to 4 carbon atoms;
_R5 is an alkylene group having 1 to 4 carbon atoms; X is an oxygen atom or -NH-;
Y is ^-COO- or _-SO3- ^;
_R6 is a hydrogen atom or a methyl group;
_R7 is a hydrogen atom or a methyl group;
R ₈ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms;
four of R ₁₆ to R ₂₀ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R ₁₆ to R ₂₀ represents CH ₂ ═C(R ₂₁ );
R ₁₅ represents an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms;
_R21 represents a hydrogen atom or a methyl group;
** represents the reaction site with the betaine agent (D).

The present invention also relates to a cell culture medium containing the cell culture medium additive.

The present invention also relates to the cell culture medium, in which the concentration of the water-soluble vinyl polymer (a) in the culture medium is 0.001 to 1% by mass.

The present invention also relates to a method for producing spheroids or biologically active substances, which comprises culturing cells in the cell culture medium.

The cell medium additive of the present invention can increase the production amount of a physiologically active substance when the additive is added to a cell medium and the obtained medium is used to culture cells capable of producing the physiologically active substance. In particular, the productivity of the physiologically active substance per amount of DNA can be increased. In addition, the addition of the cell medium additive of the present invention can promote the productivity of the desired physiologically active substance, which is also useful in terms of reducing production costs and labor. For example, in the case of antibodies, it can greatly contribute to the mass supply of biopharmaceuticals such as antibody drugs. In the case of drug-metabolizing enzymes, it can greatly contribute to drug discovery screening in pharmaceutical development.
In addition, the cell culture medium additive of the present invention can be added to a cell culture medium, and the obtained medium can be used to obtain cells with excellent cell activity and spheroid formation. When cell culture is performed using the cell culture medium additive, the cell function can be improved, and therefore the additive can be used in applications such as drug discovery screening.

The cell culture medium additive of the present invention is characterized by containing a water-soluble vinyl polymer (a).

<Water-soluble vinyl polymer (a)>
In the present invention, water solubility refers to the fact that the resin can be completely dissolved in water without separation or precipitation after 1 g of the resin is placed in 99 g of ion-exchanged water at 25° C., stirred, and left at 25° C. for 24 hours. The betaine structure refers to a structure in which a positive charge and a negative charge are not adjacent to each other in the same molecule, and a dissociable hydrogen atom is not bonded to the positively charged atom. The water-soluble vinyl polymer (a) refers to a water-soluble vinyl polymer having this betaine structure in its molecular structure. By having a hydrophilic betaine structure in the molecule, the electrostatic interaction with cells can be controlled, and the environment around the cells can be appropriately controlled, and for example, the antibody productivity of antibody-producing cells and the proliferation of established cell lines can be improved.

The water-soluble vinyl polymer (a) has at least any one of the structural units represented by the following general formulas 1 to 3 in its side chain. The structural units are preferably contained in an amount of 30 to 95% by mass, and more preferably 40 to 80% by mass, relative to 100% by mass of the total of the monomer units constituting the water-soluble vinyl polymer (a). When in the above range, the polarity is appropriately controlled, and the cellular performance, including the cellular antibody productivity, can be further improved.

(Wherein,
_R2 is an alkylene group having 1 to 6 carbon atoms;
R ₃ and R ₄ each independently represent an alkyl group having 1 to 4 carbon atoms;
_R5 is an alkylene group having 1 to 4 carbon atoms; X is an oxygen atom or -NH-;
Y is ^-COO- or _-SO3- ^;
_R7 is a hydrogen atom or a methyl group;
R ₈ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms;
four of R ₁₀ to R ₁₄ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, one of R ₁₀ to R ₁₄ represents a bonding position with the main chain of a vinyl polymer;
R ₁₅ represents an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms;
* indicates the bonding position with the main chain of the vinyl polymer.)

Such a water-soluble vinyl polymer (a) is preferably obtained by the following method.
That is,
(1) At least one of the monomers (A1) to (A3) represented by the following general formulas 4 to 6 is copolymerized with other monomers as required. The resulting copolymer is called a water-soluble vinyl polymer (a1).
or,
(2) At least one of the monomers (A4) to (A6) represented by the following general formulas 7 to 9 is copolymerized with other monomers as required, and the moiety derived from the monomers (A4) to (A6) in the obtained copolymer is reacted with a betaining agent (D) described below. The obtained reaction product has a betaine structure like the copolymer using the monomer (A1), and is called a water-soluble vinyl polymer (a2).

(Wherein,
_R1 is a hydrogen atom or a methyl group;
_R6 is a hydrogen atom or a methyl group;
_R9 is a hydrogen atom or a methyl group;
four of R ₁₆ to R ₂₀ represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and one of R ₁₆ to R ₂₀ represents CH ₂ ═C(R ₂₁ );
_R21 represents a hydrogen atom or a methyl group;
** represents the reaction site with the betaine agent (D).
The other symbols are the same as those in formulas 1 to 3.

<Water-soluble vinyl polymer (a1)>
As described above, the water-soluble vinyl polymer (a1) contains, as a copolymer structural unit, at least any one of the monomers (A1) to (A3) selected from the group represented by the general formulas 4 to 6. By using the monomers (A1) to (A3), a betaine structure can be introduced into the side chain of the vinyl polymer.

<Monomer (A1)>
As shown in general formula 4, the monomer (A1) has one ethylenically unsaturated group and a betaine structure in one molecule.
Examples of such monomers include N-(meth)acryloyloxymethyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxymethyl-N,N-diethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxyethyl-N,N-diethyl ... N-(meth)acryloyloxyalkyl-N,N-dialkylammonium alkyl-α-carboxybetaine such as N-(meth)acryloyloxybutyl-N,N-diethylammonium methyl-α-carboxybetaine; N-(meth)acrylamidoalkyl-N,N-dialkylammonium alkyl-α-carboxybetaine such as N-(meth)acrylamidopropyl-N,N-dimethylammonium methyl-α-carboxybetaine and N-(meth)acrylamidopropyl-N,N-diethylammonium methyl-α-carboxybetaine; N-(meth)acrylamidoalkyl-N,N-dialkylammonium alkyl-α-carboxybetaine such as N-(meth)acrylamidopropyl-N,N-dimethylammonium methyl-α-carboxybetaine; N-(meth)acrylamidoalkyl-N,N-dialkylammonium alkyl-α-carboxybetaine, such as N-(meth)acrylamidopropyl-N,N-diethylammonium methyl-α-carboxybetaine; N-(meth)acryloyloxymethyl-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxymethyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxymethyl-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxymethyl-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxyethyl ... N-(meth)acryloyloxyethyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium propyl ...propyl-α-sulfobetaine N-(meth)acryloyloxyalkyl-N,N-dimethylammonium alkyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium butyl-α-sulfobetaine, etc.; N-(meth)acryloyloxyalkyl-N,N-dimethylammonium alkyl-α-sulfobetaine; N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxy Dimethoxymethoxy-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-di Methylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium Examples of such sulfobetaines include N-(meth)acryloyloxyalkoxyanelkoxy-N,N-dimethylammonium alkyl-α-sulfobetaines such as ethyl-α-sulfobetaine, N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium propyl-α-sulfobetaine, and N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium butyl-α-sulfobetaine; and N-(meth)acrylamidoalkyl-N,N-dialkylammonium alkyl-α-sulfobetaines such as N-(meth)acrylamidopropyl-N,N-dimethylammonium propyl-α-sulfobetaine and N-(meth)acrylamidopropyl-N,N-dimethylammonium butyl-α-sulfobetaine. From the viewpoint of the productivity of physiologically active substances, sulfobetaine is preferred. In the present invention, the term (meth)acryl indicates methacryl or acrylic.

<Monomer (A2)>
As shown in general formula 5, the monomer (A2) also has one ethylenically unsaturated group and a betaine structure in one molecule.
Examples of such monomers include 1-vinyl-2-alkyl-3-(4-sulfoalkyl)imidazolium inner salts such as 1-vinyl-3-(3-sulfopropyl)imidazolium inner salt, 1-vinyl-3-(3-sulfobutyl)imidazolium inner salt, 1-vinyl-2-methyl-3-(3-sulfopropyl)imidazolium inner salt, and 1-vinyl-2-methyl-3-(4-sulfobutyl)imidazolium inner salt.

<Monomer (A3)>
As shown in general formula 6, the monomer (A3) also has one ethylenically unsaturated group and a betaine structure in one molecule.
Examples of such monomers include 2-vinyl-1-(3-sulfoalkyl)pyridinium inner salts such as 2-vinyl-1-(3-sulfopropyl)pyridinium inner salt and 2-vinyl-1-(3-sulfobutyl)pyridinium inner salt; and 4-vinyl-1-(3-sulfoalkyl)pyridinium inner salts such as 4-vinyl-1-(3-sulfopropyl)pyridinium inner salt and 4-vinyl-1-(3-sulfobutyl)pyridinium inner salt.

<Water-soluble vinyl polymer (a2)>
As described above, the water-soluble vinyl polymer (a) in the present invention does not necessarily have to be a copolymer obtained by copolymerizing the monomers (A1) to (A3) themselves, but can be obtained through the following steps: It is possible.
That is, at least one of the monomers (A4) to (A6) represented by the general formulas 7 to 9, which can be called the precursors of the monomers (A1) to (A3), and other monomers as required, The water-soluble vinyl polymer can be obtained by copolymerizing the above-mentioned compound and reacting at least a part of the nitrogens represented by ** in the obtained copolymer with a betaining agent (D). (a2) has a betaine structure in the side chain, similar to the copolymer using the monomers (A4) to (A6). It is preferable to use the above in such a manner that all of the above is converted to betaine.

Examples of such monomers (A4) include:
N-(meth)acryloyloxymethyl-N,N-dimethylamine,
N-(meth)acryloyloxyethyl-N,N-dimethylamine,
N-(meth)acryloyloxypropyl-N,N-dimethylamine,
N-(meth)acryloyloxybutyl-N,N-dimethylamine,
N-(meth)acryloyloxymethyl-N,N-diethylamine,
N-(meth)acryloyloxyethyl-N,N-diethylamine,
N-(meth)acryloyloxypropyl-N,N-diethylamine,
N-(meth)acryloyloxybutyl-N,N-diethylamine,
N-(meth)acryloyloxyalkyl-N,N-dialkylamines such as;
N-(meth)acrylamidopropyl-N,N-dimethylamine,
N-(meth)acrylamidopropyl-N,N-diethylamine,
N-(meth)acrylamidoalkyl-N,N-dialkylamines such as;
N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylamine,
N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylamine,
N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylamine,
N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylamine,
and N-(meth)acryloyloxyalkoxyanelkoxy-N,N-dimethylamines such as these.

Examples of such monomers (A5) include:
1-vinylimidazole, 1-vinyl-2-methyl-imidazole,
Examples of 1-vinyl-2-alkyl-imidazoles include those mentioned above.

Examples of such monomers (A6) include vinylpyridines such as 4-vinyl-pyridine and 2-vinyl-pyridine.

<Betaining Agent (D)>
The betaining agent (D) is selected from the group consisting of cyclic sulfonate esters (D1), ω-halogenated alkylsulfonate metal salts (D2), cyclic carboxylate esters (D3) and ω-halogenated alkylcarboxylate metal salts (D4). This is a group of compounds used to sulfobetaine or carbobetaine the nitrogen represented by ** in the monomers (A4) to (A6) represented by the general formulae 7 to 9 after polymerization.

Examples of such cyclic sulfonic acid esters (D1) include 1,2-ethane sultone, 1,3-propane sultone, and 1,4-butane sultone.

Examples of such ω-halogenated alkylsulfonic acid metal salts (D2) include sodium 2-chloroethanesulfonate, sodium 2-bromoethanesulfonate, sodium 3-chloropropanesulfonate, sodium 3-bromopropanesulfonate, sodium 4-chlorobutanesulfonate, and sodium 4-bromobutanesulfonate.

Examples of such cyclic carboxylic acid esters (D3) include β-propiolactone, γ-butyrolactone, and δ-valerolactone.

Examples of such ω-halogenated alkyl carboxylate metal salts (D4) include sodium 2-chloroacetate, sodium 2-bromoacetate, sodium 3-chloropropionate, sodium 3-bromopropionate, sodium 4-chlorobutyrate, sodium 4-bromobutyrate, sodium 5-chloropentanoate, and sodium 5-bromopentanoate.

<Monomer (B)>
When obtaining the water-soluble vinyl polymers (a1) and (a2), in addition to the monomers (A1) to (A3) and (A4) to (A6), a monomer (B) having a partition coefficient LogP of 0.8 to 10 can be used, and it is preferable that the water-soluble vinyl polymer (a) contains a structural unit based on the monomer (B). By introducing a structural unit based on the monomer (B), the polarity is appropriately controlled, the hydrophobic interaction with cells is controlled, and effects including the antibody productivity of cells can be maximized.

Examples of the monomer (B) having a partition coefficient Log P of 0.8 to 10 include:
Alkyl ester (meth)acrylates such as methyl methacrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-butyl (meth)acrylate, pentyl (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, dodecyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, isostearyl (meth)acrylate, behenyl (meth)acrylate, cyclohexyl (meth)acrylate, and isobornyl (meth)acrylate;
Aromatic ester (meth)acrylates such as phenyl (meth)acrylate, benzyl (meth)acrylate, and phenoxyethyl (meth)acrylate;
Examples of the aromatic vinyl monomer include styrene, α-methylstyrene, 2-methylstyrene, chlorostyrene, allylbenzene, and ethynylbenzene.
These may be used alone or in combination of two or more kinds.

The partition coefficient LogP is a numerical value that indicates the properties of a chemical substance, and is a constant value that is independent of the amount added. It is the common logarithm of the concentration of each phase when the substance in question is in equilibrium in a mixture of water and 1-octanol, where the aqueous phase and the octanol phase are in contact with each other. As LogP increases, there is a tendency for the substance to be relatively more hydrophobic, and as LogP decreases, there is a tendency for the substance to be relatively more hydrophilic.

LogP can generally be measured by the flask shaking method described in JIS Z7260-107 (2000). Instead of actual measurement, LogP can also be estimated by computational chemistry methods or empirical methods. Known methods and software can be used to calculate LogP, but in the present invention, LogP is calculated using a program built into CambridgeSoft's system: ChemdrawPro11.0.

The structural units based on monomer (B) preferably account for 5 to 70% by mass, more preferably 20 to 60% by mass, of the total 100% by mass of the monomer units constituting the water-soluble vinyl polymer (a). By making it 70% by mass or less, hydrophobic interactions with cells can be suppressed. By making it 5% by mass or more, the amount of monomers (A1) to (A3) or (A4) to (A6) becomes relatively small, and the water solubility of the water-soluble vinyl polymer (a) can be appropriately suppressed.

<Monomer (C)>
In obtaining the water-soluble vinyl polymers (a1) and (a2), in addition to the monomers (A1) to (A3), (A4) to (A6), and (B), a monomer (C) having a partition coefficient Log P outside the range of 0.8 to 10 can also be used.
Examples of the monomer (C) having a partition coefficient LogP outside the range of 0.8 to 10 include:
Hydroxyl group-containing monomers such as 2-hydroxyethyl (meth)acrylate and 2-hydroxypropyl (meth)acrylate;
Carboxylic acid-containing monomers such as acrylic acid, methacrylic acid, itaconic acid, etc.;
Amide group-containing monomers such as acrylamide, methacrylamide, and N,N-dimethylacrylamide;
Sulfonic acid group-containing monomers such as vinyl sulfonic acid and styrene sulfonic acid;
Monomers containing a polyethylene glycol chain, such as polyethylene glycol (meth)acrylate and methoxypolyethylene glycol (meth)acrylate;
Examples of such monomers include monomers having polar functional groups, such as phosphate group-containing monomers, such as (2-hydroxyethyl) methacrylate acid phosphate.

In the present invention, the ethylenically unsaturated bond group of the monomer (C) is preferably a (meth)acrylate group or an aromatic vinyl group from the viewpoint of polymerizability.

When the other monomer (C) is used, the content thereof is preferably 0 to 65 mass%, and more preferably 0 to 40 mass%, in 100 mass% in total of the monomer units constituting the water-soluble vinyl polymer (a).
When a monomer having a polar functional group is used as the other monomer (C), the content thereof is preferably 0 to 20 mass% in 100 mass% of the total monomer units constituting the water-soluble vinyl polymer (a).
The amount of the monomer (C) is preferably small so as not to inhibit the functional expression of the betaine structure formed by reacting the portion derived from the monomers (A1) to (A3) or (A4) to (A6) with the betaine agent (D).

<Cell Culture Media>
As the cell culture medium to which the water-soluble vinyl polymer (a) is added, a conventionally known cell culture medium can be used, for example, various commercially available media (αMEM, MEM, DMEM, IMDMEM, RPMI1640, DMEM/F12, etc.) or combinations thereof.
The concentration of the water-soluble vinyl polymer (A), which is an additive for a cell culture medium, in the culture medium is preferably 0.001 to 1 mass %, more preferably 0.01 to 1 mass %, and further preferably 0.1 to 1 mass %.
It is preferable to add various growth factors (e.g., epidermal growth factor, insulin-like growth factor, nerve growth factor, hepatocyte growth factor, vascular endothelial growth factor, basic fibroblast growth factor, transferrin, steroid hormones, 2-mercaptoethanol, etc.), various animal sera (e.g., fetal bovine serum (FBS), bovine serum, etc.), serum substitutes, etc. to the cell culture medium as necessary.

<Method of manufacturing physiologically active substances and spheroids>
By culturing cells in the cell culture medium of the present invention, a physiologically active substance can be produced with high efficiency. Furthermore, by culturing cells in the cell culture medium of the present invention, spheroids can be formed.

<Biologically active substances>
The physiologically active substance according to the present embodiment is, for example, an antibody protein (hereinafter referred to as an antibody), albumin, a drug-metabolizing enzyme, etc. The antibody produced here is not particularly limited, and is, for example, a mouse monoclonal antibody, a humanized monoclonal antibody, or a human monoclonal antibody. The immunoglobulin class is not particularly limited, and is, for example, IgG (for example, IgG1, IgG2, etc.). The drug-metabolizing enzyme produced is not particularly limited, and is, for example, alcohol dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, superoxide dismutase, monoamine oxidase, diamine oxidase, epoxide hydrolase, esterase, amidase, UDP-glucuronosyltransferase, glutathione S-transferase, γ-glutamyltranspeptidase, acetyltransferase, sulfotransferase, cytochrome P450, etc.
In the culture method or production method of the present invention, a vessel or device usually used for culture can be used for culture, such as a multi-well plate, a petri dish, a culture flask, a spinner flask, a jar fermenter, a fermenter, a roller bottle, a hollow fiber, a microcarrier, etc.

<Cells>
The cells to be cultured by the culture method of the present invention are not particularly limited as long as they are cells that can be used to produce physiologically active substances, and examples of cells capable of producing antibodies include CHO cells, BHK cells, HepG2 cells, rodentmyeloma cells (mouse myeloma cells such as SP2/O cells and NSO cells), hybridomas, insect cells, and transformed cells into which foreign genes have been introduced, and hybridomas obtained by cell fusion of CHO cells, SP2/O cells, NSO cells, etc. may also be used. Examples of cells capable of producing drug-metabolizing enzymes include primary hepatocytes collected from the liver of an animal, hepatocytes induced to differentiate from stem cells such as ES cells, iPS cells, and mesenchymal stem cells, and cell lines derived from liver cancer such as HepG2 cells and HuH-7 cells.

The culture conditions in this embodiment may be the same as those for ordinary animal cell culture conditions, for example, a 5% by volume _CO2 atmosphere at a temperature of 37°C.

To collect cells from the culture medium, in the case of suspension cells, for example, the culture medium is directly put into a centrifuge or a filter to collect the cells. In the case of adherent cells, for example, a 0.25% trypsin-0.02% EDTA solution is added to suspend the cells, and then the cells are collected by centrifugation or filtration.

When a physiologically active substance, particularly an antibody, is produced by cell culture and accumulates in the culture medium, the supernatant is obtained by filtration or centrifugation and then harvested from this. In the case of a substance that accumulates within cells, the cells obtained by filtration or centrifugation are homogenized, sonicated, or treated with chemicals to obtain a supernatant from which the product has been extracted.

To separate and purify the antibody from the above supernatant, a suitable combination of known methods is used. For example, ammonium sulfate precipitation, dialysis, ultrafiltration, electrophoresis, gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc. are preferred.

The present invention will be described in more detail below with reference to examples, but the following examples are not intended to limit the scope of the invention. In the examples and comparative examples, "parts" refers to "parts by mass", "mol" refers to the amount of substance, and "mass%" refers to the proportion of the amount of substance in the total monomers.

<Synthesis of various betaine monomers>
N-methacryloyloxyethyl-N,N-dimethylammonium butyl-α-sulfobetaine used in the synthesis of water-soluble vinyl polymer (a) was synthesized with reference to Japanese Patent No. 5690645. Similarly, N-methacryloyloxyethyl-N,N-dimethylammonium methyl-α-carbobetaine was synthesized with reference to Japanese Patent No. 3878315, and 1-vinyl-3-(3-sulfopropyl)imidazolium inner salt and 2-vinyl-1-(3-sulfopropyl)pyridinium inner salt were synthesized with reference to Japanese Patent No. 3584998.

[Production Example 1]
<Preparation of Water-Soluble Vinyl Polymer (a)>
A reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux condenser was charged with 150 parts of ethanol, and the internal temperature was raised to 75° C. and thoroughly substituted with nitrogen. Separately prepared 0.4 parts of 2,2′-azobis-2-methylpropionavidin and 100 parts (100% by mass) of N-methacryloyloxyethyl-N,N-dimethylammonium butyl-α-sulfobetaine as monomer (A1) were added dropwise for 3 hours while maintaining the internal temperature at 75° C., and stirring was continued for another 2 hours. After confirming that the conversion rate exceeded 98% by measuring the solid content, the mixture was cooled and taken out. Thereafter, the ethanol was completely evaporated in an oven to obtain a water-soluble vinyl polymer (a).
In addition, 1 g of the obtained vinyl polymer (a) was added to 99 g of ion-exchanged water at 25° C., stirred and dissolved, and then allowed to stand for 24 hours at 25° C. As a result, neither separation nor precipitation was observed in these resins, indicating that they were completely soluble and water-soluble.

<Preparation of cell culture medium additives>
The water-soluble vinyl polymer (a) from which the solvent had been evaporated was dissolved in phosphate buffered saline (hereinafter referred to as PBS solution) to obtain a cell culture medium additive of Production Example 1 having a concentration of 10% by mass.

[Production Examples 2 to 13]
Using the formulation shown in Table 1, a water-soluble vinyl polymer (a) was synthesized in the same manner as in Production Example 1, and dissolved in a PBS solution to obtain cell culture medium additives of Production Examples 2 to 13.

The symbols in the table are as follows:
DMBS: N-methacryloyloxyethyl-N,N-dimethylammonium butyl-α-sulfobetaine DMMC: N-methacryloyloxyethyl-N,N-dimethylammonium methyl-α-carbobetaine VSPI: 1-vinyl-3-(3-sulfopropyl)imidazolium inner salt VSPP: 2-vinyl-1-(3-sulfopropyl)pyridinium inner salt BMA: butyl methacrylate St: styrene HEA: 2-hydroxyethyl acrylate DEAA: diethylacrylamide

[Production Example 14]
A reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux condenser was charged with 150 parts of methyl ethyl ketone, the internal temperature was raised to 75° C., and the atmosphere was sufficiently replaced with nitrogen. A mixture of 0.4 parts of 2,2′-azobis-2-methylpropionavidin, 60 parts (60% by mass) of 2-(dimethylamino)ethyl methacrylate as monomer (A4), 30 parts (30% by mass) of butyl methacrylate as monomer (B), and 10 parts (10% by mass) of 2-hydroxyethyl acrylate as monomer (C), which had been prepared separately, was added dropwise for 3 hours while maintaining the internal temperature at 75° C., and stirring was continued for another 2 hours to obtain a copolymer.
After confirming that the conversion rate exceeded 98% by solid content measurement, 47 parts of 1,3-propane sultone (equimolar amount to the monomer (A4)) as a betaining agent (D), 50 parts of ethanol, and 200 parts of ion-exchanged water were added, and stirring was continued for a further 20 hours while distilling off methyl ethyl ketone and ethanol.
As shown in the reaction formula below, a ring-opening reaction of 1,4-butane sultone can result in a vinyl polymer having a structure derived from N-methacryloyloxyethyl-N,N-dimethylammoniumbutyl-α-sulfobetaine, which is one type of monomer (A1), in its side chain.

The mixture was then cooled, taken out, and placed in an oven to completely evaporate the solvent.
In addition, 1 g of the obtained vinyl polymer (a) was added to 99 g of ion-exchanged water at 25° C., stirred and dissolved, and then allowed to stand for 24 hours at 25° C. As a result, neither separation nor precipitation was observed in these resins, indicating that they were completely soluble and water-soluble.

<Preparation of cell culture medium additives>
The water-soluble vinyl polymer (a) from which the solvent had been evaporated was dissolved in phosphate buffered saline (hereinafter referred to as PBS solution) to obtain a cell culture medium additive of Production Example 14 having a concentration of 10% by mass.

[Production Examples 15 to 19]
A vinyl polymer was obtained with the composition shown in Table 2, and the moiety derived from the monomers (A4) to (A6) in the vinyl polymer was reacted with a betaining agent (D) to synthesize a water-soluble vinyl polymer (a), which was then dissolved in a PBS solution to obtain the cell culture medium additives of Production Examples 15 to 19. The reaction formulae between the moiety derived from the monomers (A4) to (A6) in the vinyl polymer and the betaining agent (D) are shown in the following formulae 11 to 15. As a result of this reaction, it is found that the water-soluble vinyl polymer (a) has a betaine structure in the side chain.

The symbols in the table are as follows:
DM: N-methacryloyloxyethyl-N,N-dimethylamine VI: 1-vinylimidazole VP: 2-vinylpyridine BS: 1,4-butane sultone PS: 1,3-propane sultone SBS: Sodium 4-bromobutanesulfonate PL: β-propiolactone SC: Sodium chloroacetate

[evaluation]
The obtained cell culture medium additive was evaluated as follows. The results are shown in Table 3.
The following (1) evaluation of cell medium additives, (2) evaluation of production amount of physiologically active substances, and (3) evaluation of cell activity and spheroid formability were performed by adding a predetermined amount of cell medium additives to each basal medium and adjusting the medium composition by pipetting, and evaluating the final concentration in the range of 0.01% by mass to 1% by mass.

(1) Evaluation of cell culture medium additives

<Cytotoxicity evaluation>
A medium was prepared by adding 10% fetal bovine serum (FBS) to a low glucose-DMEM medium (Sigma-Aldrich Co., Ltd.) (hereinafter, the above medium is referred to as 10% FBS-DMEM medium). A cell medium additive containing a water-soluble vinyl polymer was added to the 10% FBS-DMEM medium to the concentration shown in Table 3, and the medium composition was prepared by stirring. Human liver cancer cell line HepG2 cells were adjusted to 100,000 cells/mL, and then seeded into the wells of a 96-well flat-bottom microplate at 100 μL per well. Then, the plate was cultured for 2 days in a stationary state in a CO ₂ incubator (37° C., 5% CO ₂ ). Then, the medium was replaced with the medium composition containing the above-mentioned cell medium additive containing a water-soluble vinyl polymer, and after culturing for 1 day, a cytotoxicity test using the WST-8 reagent was evaluated. After adding 10 μL of WST-8 reagent (Dojin Laboratories, Inc.) to the culture solution containing the cultured hepatocytes, the cells were allowed to stand in a _CO2 incubator (37° C., 5% _CO2 ) for 2 hours. After 2 hours, the absorbance at 450 nm was measured using a microplate reader, and the cytotoxicity was evaluated by subtracting the absorbance of the culture solution to which the WST-8 reagent had not been added. The cytotoxicity was evaluated as a relative value, with the result of culturing without adding the cell medium additive containing a water-soluble vinyl polymer being taken as 1. The evaluation results are shown in Table 3.
◎: 1.0 or more (very good)
○: 0.90 or more to less than 1.0 (good)
×: Less than 0.90 (poor)

(2) Evaluation of production volume of biologically active substances

<Evaluation of antibody productivity>
The medium used was Dynamis Medium (Gibco), and L-Glutamin was added to 1.0% by mass. Hereinafter, this is referred to as the basal medium. Two sets of this were prepared, and the obtained cell medium additive containing a water-soluble vinyl polymer was added to the concentration shown in Table 3. An IgG gene was introduced into two types of medium containing a different concentration of a cell medium additive containing a water-soluble vinyl polymer, and a CHO cell line (ATCC CRL-12455) that secretes and produces IgG antibodies was added to each of the two types of medium, and a cell suspension was prepared with a concentration of the CHO cell line of 1,000,000 cells/mL. 20 mL of the obtained cell suspension was seeded in a 125 mL Erlenmeyer flask and cultured at 37°C. During the culture, 15 mL of the supernatant was collected once every 3 to 4 days before the nutrient source was depleted, and replaced with 15 mL of new basal medium, and this was repeated five times. The cell culture solution after the medium exchange was repeated five times was centrifuged to recover the medium supernatant. The amount of antibody in the medium was measured using a Human IgG ELISA Quantitation Set manufactured by Bethyl Laboratories, Inc. The amount of DNA in the precipitated cell group was also quantified and calculated as the amount of antibody produced per unit amount of DNA. The results were evaluated as a relative value when the results when the cell culture medium additive containing a water-soluble vinyl polymer was not added were taken as 1. The evaluation results are shown in Table 3.
◎: 1.2 or more (very good)
○: 1 or more to less than 1.2 (good)
×: Less than 1 (defective)

<Albumin production evaluation>
A medium composition was prepared by adding a cell medium additive containing a water-soluble vinyl polymer to a 10% FBS-DMEM medium to the concentration shown in Table 3 and stirring. Human hepatoma cell line HepG2 cells were seeded with the medium composition containing the cell medium additive containing the water-soluble vinyl polymer so as to have a concentration of 250,000 cells/mL, and then dispensed into wells of a 96-well U-bottom microplate at 200 μL per well. The plate was then cultured for 3 days in a stationary state in a CO ₂ incubator (37° C., 5% CO ₂ ). The culture solution containing hepatocytes after 3 days of culture was collected and centrifuged to collect the medium supernatant. The amount of albumin in the medium was measured using an Albumin ELISA Quantitation Set manufactured by Bethyl Laboratories, Inc. The amount of DNA in the precipitated cell group was also quantified and calculated as the amount of albumin produced per unit amount of DNA. The results were evaluated as a relative value, with the result of culturing without adding the water-soluble vinyl polymer-containing cell culture medium additive being taken as 1. The evaluation results are shown in Table 3.
◎: 1.2 or more (very good)
○: 1 or more to less than 1.2 (good)
×: Less than 1 (defective)

<Evaluation of drug metabolizing enzyme activity>
A cell medium additive containing a polymer was added to a 10% FBS-DMEM medium to the concentration shown in Table 3, and the medium composition was prepared by stirring. Human liver cancer cell line HepG2 cells were seeded with the medium composition containing the cell medium additive containing the water-soluble vinyl polymer so as to have a concentration of 250,000 cells/mL, and then dispensed into wells of a 96-well U-bottom microplate at 200 μL per well. The plate was then cultured in a stationary state in a CO2 incubator (37°C, 5% CO2) for 3 days. After culturing for 3 days, the activity of drug metabolizing enzymes was evaluated. Drug metabolizing enzyme activity was evaluated using P450-GloTM CYP3A4 Assay Kits (manufactured by Promega), and Luciferin-IPA was used as the substrate. The medium was replaced with a 10% FBS-DMEM medium containing the substrate, and incubated for 4 hours. The medium after incubation was transferred to a black plate for luminescence measurement, a detection reagent (Luciferindetection reagent) was added, and the plate was left to stand at room temperature for 20 minutes. The amount of luminescence from this plate was measured using a plate reader (manufactured by Berthold). The amount of DNA in the cell population was also quantified and calculated as the drug metabolizing enzyme activity per unit amount of DNA. The results were evaluated as a relative value when the results were obtained without adding a cell medium additive containing a water-soluble vinyl polymer, which was set to 1. The evaluation results are shown in Table 3.
◎: 1.2 or more (very good)
○: 1.0 or more and less than 1.2 (good)
×: Less than 1 (defective)

<Gene expression analysis by qRT-PCR method>
A cell medium additive containing a polymer was added to a 10% FBS-DMEM medium to the concentration shown in Table 3, and the medium composition was prepared by stirring. After seeding the human hepatoma cell line HepG2 cells with the medium composition containing the cell medium additive containing the water-soluble vinyl polymer so as to give a concentration of 250,000 cells/mL, the medium composition was dispensed into the wells of a 96-well U-bottom microplate so that 200 μL per well was dispensed. Then, the plate was cultured for 3 days in a stationary state in a CO2 incubator (37 ° C, 5% CO2). After 3 days of culture, the culture solution containing the hepatocytes was collected, and the cells were collected by centrifugation. Total RNA was extracted from the collected HepG2 cells using RNeasy Micro Kit (manufactured by QIAGEN). cDNA was synthesized by reverse transcription reaction using the extracted total RNA using ReverTra Ace qPCR RT Master Mix (manufactured by TOYOBO). qRT-PCR was performed using TaqMan probes (Life Technologies, CYP3A4: Hs00604506_m1, Albumin: Hs00609411_m1) of cDNA and the target genes (CYP3A4 and Albumin). GAPDH (Life Technologies, Hs02786624_g1) was used as the reference gene. The relative expression level of each gene was calculated using the comparative Ct method with GAPDH as the standard. The results were determined as a relative value when the results were obtained without adding a cell medium additive containing a water-soluble vinyl polymer, which was taken as 1. The evaluation results are shown in Table 3.
◎: 2 or more (very good)
○: 1 or more and less than 2 (good)
×: Less than 1 (defective)

(3) Evaluation of cell activity and spheroid formation

<ATP activity>
A medium composition was prepared by adding a cell medium additive containing a water-soluble vinyl polymer to a 10% FBS-DMEM medium to the concentration shown in Table 3 and stirring. Human liver cancer cell line HepG2 cells were seeded with the medium composition containing the cell medium additive containing a water-soluble vinyl polymer to give a concentration of 100,000 cells/mL, and then dispensed into wells of a 96-well U-bottom microplate at 100 μL per well. The plate was then cultured stationary for 5 days in a _CO2 incubator (37°C, 5% _CO2 ).
ATP activity was evaluated by performing an ATP assay after 1 and 5 days. Specifically, 100 μL of ATP reagent ("clump" ATP measurement reagent: manufactured by Toyo B-Net Co., Ltd.) was added to the wells after incubation, pipetted 5 times, and left at room temperature for 5 minutes, after which 100 μL of the reagent/cell lysate was dispensed onto a separate plate and stirred for 1 minute. The amount of luminescence was measured using Mithras LB940 (manufactured by Berthold Co., Ltd.). The evaluation results are shown in Table 3.
ATP activity=(luminescence amount after 5 days of culture)/(luminescence amount after 1 day of culture)×100
◎: 120% or less ATP activity, ATP activity is quite high (very good)
◯: 50%≦ATP activity<120% ATP activity is high (good)
△: 20%≦ATP activity<50% ATP activity is slightly high (passable)
×: ATP activity < 20% ATP activity is low (poor)

<Spheroid formation>
A medium composition was prepared by adding a cell medium additive containing a water-soluble vinyl polymer to a 10% FBS-DMEM medium to the concentration shown in Table 3 and stirring. A human liver cancer cell line (HepG2 cells) was seeded with the medium composition containing the cell medium additive containing a water-soluble vinyl polymer so as to give a concentration of 100,000 cells/mL, and then dispensed into wells of a microplate (Sumitomo Berakuit Co., Ltd. PrimeSurfaceMS-9096U) at 100 μL per well. The plate was then cultured stationary for 3 days in a CO2 incubator (37°C, 5% CO2).
The spheroid forming ability was evaluated by photographing the cell culture state after 3 days at 40x magnification using a transmission optical microscope and observing the cell morphology according to the following criteria. The evaluation results are shown in Table 3.
〇: One spheroid formed (good)
△: Multiple spheroids formed (slightly poor)
×: No spheroids formed (defective)

<Results>
From the results in Table 3, it was found that the cell culture medium additives of Examples 1 to 19 had low cytotoxicity and exhibited excellent antibody productivity, albumin productivity, and drug metabolizing enzyme activity. On the other hand, the additives of Comparative Examples 1 to 4 were inferior to the cell culture additives of Examples 1 to 19 in terms of cytotoxicity, antibody productivity, albumin productivity, and ATP activity.

The cell culture medium additive of the present invention can increase the production amount of a physiologically active substance by adding it to a cell culture medium.
In addition, the use of the cell culture medium additive of the present invention is useful in that it can promote the productivity of a desired physiologically active substance and reduce production costs and labor. For example, it can greatly contribute to the mass supply of biopharmaceuticals such as antibody drugs.

Claims (5)

A cell culture medium additive comprising a water-soluble vinyl polymer (a) having a structural unit represented by the following general formula 1 in a side chain (excluding polymers having a polyoxyalkylene group in a side chain, the end of which is a repeating unit composed of an alkyl group having 5 to 30 carbon atoms, an alkanoyl group having 5 to 30 carbon atoms, or an aryl group having 6 to 30 carbon atoms) :
The water-soluble vinyl polymer (a) contains 30 to 100% by mass of a structural unit represented by the following general formula 1, based on 100% by mass of the total of monomer units constituting the water-soluble vinyl polymer (a):
The cell culture medium additive, characterized in that the water-soluble vinyl polymer (a) is the following (a1) or (a2):
(a1) A copolymer of monomers including a monomer (A1) represented by the following general formula 4:
(a2) A reaction product of a copolymer of a monomer containing a monomer (A4) represented by the following general formula 7 and one or more betaining agents (D) selected from the group consisting of a cyclic sulfonate ester (D1), an ω-halogenated alkylsulfonate metal salt (D2), a cyclic carboxylate ester (D3) and an ω-halogenated alkylcarboxylate metal salt (D4).

(In General Formula 1 ,
_R2 is an alkylene group having 1 to 6 carbon atoms;
R ₃ and R ₄ each independently represent an alkyl group having 1 to 4 carbon atoms;
_R5 is an alkylene group having 1 to 4 carbon atoms ;
X is an oxygen atom or -NH-,
Y represents —COO ^— or —SO ₃ ^— ;
* indicates the bonding position with the main chain of the vinyl polymer.)


(In general formulas 4 and 7,
R1 is a hydrogen atom or a methyl group _;
R2 is an alkylene group having 1 to 6 carbon atoms _;
R ₃ and R ₄ each independently represent an alkyl group having 1 to 4 carbon atoms;
R5 is an alkylene group having 1 to 4 carbon atoms _;
X is an oxygen atom or -NH-,
Y represents —COO ^— or —SO ₃ ^— ;
** represents the reaction site with the betaine agent (D). The cell culture medium additive according to claim 1, wherein the water-soluble vinyl polymer (a) contains 30 to 95% by mass of the structural unit represented by general formula 1 relative to 100% by mass of the total of the monomer units constituting the water-soluble vinyl polymer (a). 3. The cell culture medium additive according to claim 1 or 2, wherein the water-soluble vinyl polymer (a) contains a structural unit based on a monomer (B) having a partition coefficient Log P of 0.8 to 10, and the structural unit based on the monomer (B) is contained in an amount of 5 to 70% by mass relative to 100% by mass of the total monomer units constituting the water-soluble vinyl polymer (a). A cell culture medium comprising the cell culture medium additive according to any one of claims 1 to 3 ,
A cell culture medium, wherein the concentration of the water-soluble vinyl polymer (a) in the medium is 0.001 to 1 mass % . A method for producing a spheroid or a physiologically active substance, comprising culturing cells in the cell culture medium according to claim 4 .