JP2021083381A - Methods for promoting cell proliferation - Google Patents

Abstract

To provide means for promoting cell proliferation, in particular, means for promoting cell proliferation in three-dimensional culture.SOLUTION: Disclosed is a method for promoting cell proliferation, which comprises carrying out three-dimensional culture of a cell line in a medium containing a specific compound or salt thereof. The cell may be: a normal cell line of a human umbilical cord venous endothelial cell, mouse embryonic fibroblast or human embryonic renal cell; cancer cell lines such as SKOV3 cell, HeLa cell, human malignant melanoma-derived cell line A375, human epithelial-like cell cancer-derived cell line A431, human gastric adenocarcinoma-derived cell line AGS, human prostate cancer-derived cell line LNCap clone FGC, human colon adenocarcinoma-derived cell line HCT116, human alveolar basal epithelial adenocarcinoma-derived cell line A549, human prostate cancer-derived cell line DU145; or stem cells such as human mesenchymal stem cell.SELECTED DRAWING: None

Description

The present invention relates to a method for promoting cell proliferation and the like, and more particularly to a method for promoting cell proliferation under 3D culture.

In recent years, experiments using cells have been carried out extremely frequently in many fields including the field of life science for the purpose of elucidating the mechanism of action of life phenomena and establishing treatment methods for diseases. For example, as an example in the field of drug discovery, there is a method of allowing a candidate compound to act on a cancer cell to be treated and screening for a compound capable of suppressing the growth of the cancer cell. In such screening, tens of thousands of candidate compounds may be screened, and in such an embodiment, it is necessary to prepare a large amount of homogeneous cells. However, cells of higher organisms such as humans require a period of about one day even if they divide relatively quickly, and cancer cells and stem cells require several months for one cell division. There are more than the above, which is a factor that hinders rapid cell procurement. Against this background, it is required to construct a means capable of promoting the proliferation of slow-dividing cells. For example, a thiol compound having a specific structure promotes the proliferation of hematopoietic progenitor cells, and a polyprenyl compound is a hepatocyte. It has been reported that the growth of the compound is promoted (Patent Documents 1 and 2). On the other hand, in the field of drug discovery screening, three-dimensional culture of cells has been attracting attention in recent years. Three-dimensional culture is a cell culture technology that plays an intermediate role between in vitro and in vivo. In 3D culture, cells can form three-dimensional structures such as spheres (also referred to as spheroids), and thus can enable more biological assays compared to normal 2D culture. Therefore, the three-dimensional culture may be able to identify a disease therapeutic compound that could not be identified by drug discovery screening using the two-dimensional culture (Non-Patent Document 1).

Special Table No. 11-504000 Re-table 2008/155920

Breslin S et al, Drug Discov Today. 2013 18 (5-6): 240-9.

An object of the present invention is to provide a means for promoting cell proliferation, particularly a means for promoting cell proliferation in a three-dimensional culture.

As a result of diligent studies on the above-mentioned problems, the present inventors have found that a specific compound has an effect of remarkably promoting the proliferation of cells under three-dimensional culture, and further research is carried out based on such findings. This has led to the completion of the present invention.
That is, the present invention is as follows.

[1] A method for promoting cell proliferation, which comprises three-dimensionally culturing cells in a medium supplemented with the compound represented by the following general formula (I) or a salt thereof.

{In the formula, R ₁ is hydrogen, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an aryl group which may have a substituent, and R ₂ is hydrogen or an aryl group which may have a substituent. It is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or R ₁ and R ₂ are together- (CH ₂ ) _n- (in the formula, n is 2, 3, 4 or 5) may be formed, where R ₃ is hydrogen, -CONH-CH ₃ or -NHCO-CH ₃ }.
[2] The method according to [1], wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines, and stem cells.
[3] The method according to [2], wherein the normal cell line is selected from the group consisting of human umbilical vein endothelial cells (HUVEC), mouse embryonic fibroblasts (C3H10T1 / 2), and human fetal kidney cells (HEK293). ..
[4] The cancer cell lines are SKOV3 cells, HeLa cells, human malignant melanoma-derived cell line A375, human epithelial-like cell cancer-derived cell line A431, human gastric adenocarcinoma-derived cell line AGS, and human prostate cancer-derived cell. One or more selected from the group consisting of the LNCap clone FGC, human colon adenocarcinoma-derived cell line HCT116, human alveolar basal epithelial adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell DU145 [2]. ] The method described.
[5] The method according to [2], wherein the stem cell is a human mesenchymal stem cell (MSC).
[6] The method according to any one of [1] to [5], wherein the compound is the compound shown below or a salt thereof.

[7] The method according to any one of [1] to [6], wherein the concentration of the compound in the medium is 0.001 to 100 μM.
[8] A three-dimensional cell culture medium additive containing the compound represented by the following general formula (I) or a salt thereof:

{In the formula, R ₁ is hydrogen, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an aryl group which may have a substituent, and R ₂ is hydrogen or an aryl group which may have a substituent. It is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or R ₁ and R ₂ are together- (CH ₂ ) _n- (in the formula, n is 2, 3, 4 or 5) may be formed, where R ₃ is hydrogen, -CONH-CH ₃ or -NHCO-CH ₃ }.
[9] The agent according to [8], wherein the cells are selected from the group consisting of normal cells, cancer cells, and stem cells.
[10] The agent according to [9], wherein the normal cell line is selected from the group consisting of human umbilical vein endothelial cells (HUVEC), mouse embryonic fibroblasts (C3H10T1 / 2), and human fetal kidney cells (HEK293). ..
[11] The cancer cells are SKOV3 cells, HeLa cells, human malignant melanoma-derived cell line A375, human epithelial-like cell cancer-derived cell line A431, human gastric adenocarcinoma-derived cell line AGS, and human prostate cancer-derived cell line. The agent according to [9], which is selected from the group consisting of LNCap clone FGC, human colon adenocarcinoma-derived cell line HCT116, human alveolar basal epithelial adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell DU145.
[12] The agent according to [9], wherein the stem cell is a human mesenchymal stem cell (MSC).
[13] The agent according to any one of [8] to [12], wherein the compound is the compound shown below or a salt thereof.

[14] A cell culture medium containing the agent according to any one of [8] to [13] and a polysaccharide or a processed product thereof.
[15] A method for producing a sphere, which comprises three-dimensionally culturing cells in a medium supplemented with the compound represented by the following general formula (I) or a salt thereof.

{In the formula, R ₁ is hydrogen, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an aryl group which may have a substituent, and R ₂ is hydrogen or an aryl group which may have a substituent. It is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or R ₁ and R ₂ are together- (CH ₂ ) _n- (in the formula, n is 2, 3, 4 or 5) may be formed, where R ₃ is hydrogen, -CONH-CH ₃ or -NHCO-CH ₃ }.

According to the present invention, the proliferation of cells under three-dimensional culture can be promoted easily and efficiently. Further, according to the present invention, the sphere can be efficiently produced.

Hereinafter, the present invention will be described in detail.

Definitions In the present specification, the "alkyl group having 1 to 6 carbon atoms" means a linear or branched alkyl group having 1 to 6 carbon atoms, and specifically, methyl, ethyl, n-propyl. , Isobutyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl and the like. .. In the present specification, n- means normal, i- means iso, sec- means secondary, and tert- means tertiary.

Examples of the "aryl group" include monocyclic, bicyclic, tricyclic and tetracyclic carbocyclic groups in which at least one ring is aromatic and each ring has 5 to 8 ring atoms. Specifically, phenyl, indenyl, naphthyl, fluorenyl and the like can be mentioned. In particular, the aryl group can be phenyl, indenyl, naphthyl, which is a ring having 6 to 10 carbon atoms.

The "alkyl group having 1 to 6 carbon atoms" and the "aryl group" may have a substituent, and examples of such a substituent include the following. In the case of an "alkyl group having 1 to 6 carbon atoms", the following (1) to (40) can be mentioned, and in the case of an "aryl group", the following (1) to (41) can be mentioned.

(1) Harogeno group,
(2) Hydroxy group,
(3) Cyano group,
(4) Nitro group,
(5) Carboxyl group,
(6) Alkenyl group (C _2-10 alkenyl group; eg, vinyl, allyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, butazienyl, hexatrienyl, and their respective isomers),
(7) Alkynyl group (C _2-10 alkynyl group; eg, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and their respective isomers),
(8) Halogenoalkyl groups (eg, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, chloromethyl, chloroethyl, dichloroethyl, and their respective isomers),
(9) Cyclic alkyl groups (which may contain heteroatoms in the ring) (eg, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyranyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl),
(10) Aryl groups (eg, phenyl, naphthyl),
(11) Heteroaryl groups (eg, pyridyl, pyridadinyl, pyrimidinyl, pyrazinyl, furyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl (eg, 1,2,3-triazolyl, 1,2,4-triazolyl), tetrazolyl, Oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, oxadiazolyl (eg 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl), thiadiazolyl (eg 1,2,3-thiazazolyl, 1 , 2,4-thiazolyl, 1,3,4-thiazolyl), benzofuryl, benzothiophenyl, indrill, isoindrill, benzoxazolyl, benzothiazolyl, benzimidazolyl, indazolyl, benzisooxazolyl, benzisothiazolyl, benzo Oxaziazolyl, benzothiazolyl, prynyl, quinolinyl, isoquinolinyl, synnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, imidazolioxazolyl, imidazoliazolyl, imidazolyl),
(12) Alkoxy groups (eg, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy, neopentyloxy) , 2-Pentyloxy, 3-Pentyloxy, n-Hexyloxy, 2-Hexyloxy),
(13) Alkylthio groups (eg, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, tert-butylthio, n-pentylthio, isopentylthio, tert-pentylthio, neopentylthio, 2-Pentylthio, 3-Pentylthio, n-hexylthio, 2-hexylthio),
(14) An alkoxy group (synonymous with (12) above) substituted with an aryl group (synonymous with (10) above),
(15) An alkylthio group (synonymous with (13) above) substituted with an aryl group (synonymous with (10) above),
(16) Alkoxy group (synonymous with (12) above) substituted with a heteroaryl group (synonymous with (11) above),
(17) An alkylthio group (synonymous with (13) above) substituted with a heteroaryl group (synonymous with (11) above),
(18) Cyclic alkyl (may contain a heteroatom in the ring) Oxy group (eg, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, tetrahydrofuranyloxy, tetrahydropyranyloxy, aziridinyloxy, azeti Dinyloxy, pyrrolidinyloxy, piperidinyloxy, morpholinyloxy),
(19) Aryloxy group (eg, a group in which an aryl group (synonymous with (10) above) is bonded to an oxygen atom),
(20) A heteroaryloxy group (eg, a group in which a heteroaryl group (synonymous with (11) above) is bonded to an oxygen atom),
(21) A halogenoalkoxy group (eg, a group in which a halogenoalkyl group (synonymous with (8) above) is bonded to an oxygen atom),
(22) A halogenoalkylthio group (eg, a group in which a halogenoalkyl group (synonymous with (8) above) is bonded to a sulfur atom),
(23) Alkoxy group substituted with a hydroxyl group (synonymous with (12) above),
(24) An alkoxy group (synonymous with (12) above) substituted with an alkoxy group (synonymous with (12) above),
(25) Amino group,
(26) Mono or di-substituted amino groups with alkyl groups,
Here, the "alkyl group" includes a C _1-6 alkyl group, and specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n. -Pentyl, isopentyl, tert-pentyl, neopentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl and the like can be mentioned.
(27) Carbamic group,
(28) Carbamoyl groups mono- or di-substituted with alkyl groups (synonymous with "alkyl group" in (26) above) (eg, methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl).
(29) Sulfamoyl group,
(30) Mono or di-substituted sulfamoyl groups with alkyl groups (synonymous with "alkyl group" in (26) above) (eg, methyl sulfamoyl, ethyl sulfamoyl, dimethyl sulfamoyl, diethyl sulfamoyl, Ethylmethylsulfamoyl),
(31) An alkanoyl group (eg, a carbonyl group in which a hydrogen atom or an alkyl group (synonymous with "alkyl group" in (26) above) is bonded to a carbon atom),
(32) Aroyl group (eg, carbonyl group in which an aryl group (synonymous with (10) above) is bonded to a carbon atom),
(33) Alkylsulfonylamino group (eg, a sulfonylamino group substituted with an alkyl group (synonymous with "alkyl group" in (26) above))
(34) Arylsulfonylamino group (eg, a sulfonylamino group substituted with an aryl group (synonymous with (10) above)),
(35) Heteroarylsulfonylamino group (eg, sulfonylamino group substituted with heteroaryl group (synonymous with (11) above)),
(36) Acylamino group (eg, amino group substituted with an acyl group),
Here, the "acyl group" is an acyl group having _{a C 1-6} alkyl group or a C _{6-10 aryl group.} Here, the "C _1-6 alkyl group" is one of the above "alkyl groups" having 1 to 6 carbon atoms, and the "C _6-10 aryl group" is the above "aryl group". Of these, those having 6 to 10 carbon atoms. Specific examples of the acyl group include an acetyl group, a propionyl group, a butyloyl group, an isobutyroyl group, a valeroyl group, an isovaleroyl group, a pivaloyl group, a hexanoyl group, an acryloyl group, a methacryloyl group, a crotonoyle group, an isocrotonoyl group, a benzoyl group and a naphthoyl group. Basics, etc.
(37) Alkoxycarbonylamino group (eg, carbonylamino group substituted with an alkoxy group (synonymous with (12) above)),
(38) Alkylsulfonyl groups (eg, sulfonyl groups substituted with alkyl groups (synonymous with "alkyl group" in (26) above)),
(39) Alkyl sulfinyl group (eg, sulfinyl group substituted with an alkyl group (synonymous with "alkyl group" in (26) above)),
(40) Alkoxycarbonyl group (eg, methoxycarbonyl group, ethoxycarbonyl group),
(41) Alkyl group (C _1-10 alkyl group; eg, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl , 2-Pentyl, 3-Pentyl, n-Hexyl, 2-Hexyl, etc.) and the like.

If there are two or more substituents, they may be the same or different.

The compound represented by the general formula (I) may be in the form of a salt. Examples of the salt of the compound represented by the general formula (I) include salts with inorganic acids such as hydrochloric acid and hydrobromic acid, acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, and oxalic acid. Examples include salts with organic acids such as succinic acid, malic acid and benzoic acid.

The compound represented by the general formula (I) may have an E-form having an E configuration and a Z-form geometric isomer having a Z configuration depending on the type of the substituent. The present invention includes these E-forms, Z-forms or mixtures containing E-forms and Z-forms in arbitrary proportions.

The compound represented by the general formula (I) can be produced by a method known per se. The compound used in the present invention can be produced, for example, by the method described in Japanese Patent No. 5258563.

The term "three-dimensional culture (3D culture)" as used herein refers to culturing cells in a three-dimensional environment using, for example, an embedding culture method, a microcarrier culture method, or a sphere culture method. means. In embedding culture, cells are embedded and fixed in a solid or semi-solid gel substrate such as Matrigel (registered trademark), Geltrex (registered trademark), agar, methyl cellulose, collagen, gelatin, fibrin, agarose, and alginate. It is a method of culturing. In the microcarrier culture method, cells are grown in a single layer on the surface of fine particles (hereinafter, also referred to as microcarriers) slightly heavier than water, and the fine particles are stirred in a culture vessel such as a flask in a suspended state. It is for culturing. In sphere culture, after the target cells form agglomerates consisting of tens to hundreds (hereinafter, also referred to as spheres or spheroids), the agglomerates are cultured in a medium by standing or shaking. The method. In addition, as a three-dimensional culture, polysaccharides such as hyaluronic acid, deacylated gellan gum, and xanthan gum or processed products thereof (eg, cellulose nanofibers, chitin nanofibers, chitosan nanofibers, etc.) are dispersed in a liquid medium. A method of culturing cells in a three-dimensional state may be used by forming an atypical three-dimensional network and maintaining the adherent cells in a floating state in a liquid medium using this as a scaffold. When 3D culture is performed using polysaccharides or their processed products, cells are trapped in the 3D network and do not precipitate. Therefore, the cells can be cultured without requiring shaking, rotation, or the like. Three-dimensional culture using a polysaccharide or a processed product thereof can be carried out by a method known per se (see, for example, WO2014 / 017513, WO2015 / 111686).

In the present specification, three-dimensional culture using a liquid medium may be referred to as suspension culture. As used herein, the term "floating" of cells refers to a state in which the cells do not adhere to the culture vessel. Furthermore, when the cells are proliferated, differentiated or maintained, the cells are dispersed in the liquid medium and are in a floating state without external pressure, vibration, shaking, rotation operation, etc. on the liquid medium composition. It is called "floating static". Culturing cells in this state is called "suspended static culture". In "floating stationary", the period for suspending cells is, but is not limited to, at least 5 minutes or longer, preferably 1 hour or longer, 24 hours or longer, 48 hours or longer, 6 days or longer, and 21 days or longer.

Whether or not floating standing is possible, for example, polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) are uniformly dispersed in the medium composition to be evaluated and allowed to stand at 25 ° C., and at least 5 It can be evaluated by observing whether or not the floating state of the cells is maintained for a minute or more (preferably 24 hours or more, 48 hours or more).

1. 1. Method for Promoting Cell Proliferation The present invention comprises a method for promoting cell proliferation, which comprises three-dimensionally culturing cells in a medium to which a compound represented by the following general formula (I) or a salt thereof is added (hereinafter, "the present invention". Provided (sometimes referred to as "methods of invention"):

{In the formula, R ₁ is hydrogen, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an aryl group which may have a substituent, and R ₂ is hydrogen or an aryl group which may have a substituent. It is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or R ₁ and R ₂ are together- (CH ₂ ) _n- (in the formula, n is 2, 3, 4 or 5) may be formed, where R ₃ is hydrogen, -CONH-CH ₃ or -NHCO-CH ₃ }.

In one aspect of the method of the present invention, in the compound represented by the general formula (I), R ₁ has an alkyl group having 1 to 6 carbon atoms, which may have a substituent (preferably, has a substituent. It is an alkyl group having 1 to 3 carbon atoms, more preferably an ethyl group substituted with a hydroxyl group), and R ₂ and R ₃ are hydrogen.

In one aspect of the method of the invention, the compound represented by the general formula (I) is a combination of R ₁ and R ₂ − (CH ₂ ) _n − (n is 2, 3, 4 or 5). It is preferably 3, 4 or 5, more preferably 3 or 5), and R ₃ is hydrogen.

In one aspect of the method of the invention, the compound represented by the general formula (I) is a combination of R ₁ and R ₂ − (CH ₂ ) _n − (n is 2, 3, 4 or 5). It is preferably 2, 3 or 4, more preferably 2), and R ₃ is -CONH-CH ₃ .

In one aspect of the method of the present invention, in the compound represented by the general formula (I), R ₁ is an aryl group which may have a substituent (preferably a phenyl group which may have a substituent). , More preferably, a phenyl group having no substituent), R ₂ is hydrogen, and R ₃ is -NHCO-CH ₃ .

In a preferred embodiment, the compounds (Compounds A to E) used in the present invention are as follows.

In the method of the present invention, the compound represented by the above general formula (I) is added to the medium. The concentration of the compound added to the medium is not particularly limited as long as the desired effect can be obtained, and may be appropriately set in consideration of the cell type and the like. For example, the lower limit of the concentration of the compound added to the medium is usually 0.001 μM or more, preferably 0.01 μM or more, more preferably 0.1 μM or more, still more preferably 0.2 μM or more, and particularly preferably 0.3 μM. It can be more than that. The upper limit of the concentration is usually 100 μM or less, preferably 50 μM or less, more preferably 15 μM or less, still more preferably 5 μM or less, and particularly preferably 3 μM or less. In one embodiment, the final concentration of the compound in the medium is 0.001-100 μM, preferably 0.01-50 μM, more preferably 0.1-15 μM, even more preferably 0.2-5 μM, particularly preferably 0.3. It can be ~ 3 μM.

The medium that can be used in the method of the present invention is not particularly limited as long as the desired effect of the present invention can be obtained, and may be appropriately determined according to various conditions such as cell type. A commercially available product may be used as the medium. Examples of commercially available media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrition Mixture F12, DMEM / F12 medium, and McCoy's 5A medium (McCoy's 5A). , Eagle's MEM (Eagle's Minimum Essential medium; EMEM), αMEM (alpha modified Eagle's Minimum Essential medium; αMEM), MEM (Minimum Essential medium), RPMI1640 medium, Iscove's modified Dulbecco's medium (Iscove's modified Dulbecco's medium; IMDM), MCDB131 medium, William's medium E, IPL41 medium, Fisher's medium, StemPro34 (manufactured by Invigen), X-VIVO 10 (manufactured by Kenbrex), X-VIVO 15 (manufactured by Kenbrex), HPGM (manufactured by Kenbrex), StemSpan H3000 (manufactured by Kenbrex) Stemcell Technology (manufactured by Stemcell Technology), StemSpanSFEM (manufactured by Stemcell Technology), StemlineII (manufactured by Sigma Aldrich), QBSF-60 (manufactured by Quality Biological), StemProhESCSFM (manufactured by Invigen), Essential8 (registered trademark) medium (manufactured by Gibco) ), MTeSR1 medium (manufactured by Stemcell Technology), mTeSR2 medium (manufactured by Stemcell Technology), Repro FF (manufactured by Reprocell), Repro FF2 (manufactured by Reprocell), StemFit (registered trademark) AK02N (manufactured by Ajinomoto), StemFit (manufactured by Ajinomoto) Registered trademark) AK03N (manufactured by Ajinomoto), PSGrow ESC / iPSC medium (manufactured by System Bioscience), NutriStem (manufactured by Biological Industries), CSTI-7 medium (manufactured by Cell Science Research Institute), MesenPRO RS medium (manufactured by Gibco), MF-Medium (registered trademark) Medium stem cell growth medium (manufactured by Toyobo Co., Ltd.), medium for mezzanine stem cells (manufactured by Promocell), Sf-900II (manufactured by Invitrogen), Examples thereof include a medium such as Opti-Pro (manufactured by Invitrogen). In addition, a three-dimensional culture medium can be prepared by adding a polysaccharide such as deacylated gellan gum to these media. As a three-dimensional culture medium containing such a polysaccharide, for example, FCeM (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) is commercially available, and this can also be used.

In addition, sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, cell growth factors, differentiation inducers, cell adhesion factors, antibodies, enzymes, cytokines, hormones, lectins. , Extracellular matrix, physiologically active substances and the like may be added to the medium depending on the purpose.

As described above, in one aspect of the method of the present invention, three-dimensional culture can be carried out by adding a polysaccharide or a processed product thereof (hereinafter, may be referred to as a polysaccharide or the like) to a medium.

The polysaccharides or processed products thereof that can be used in the method of the present invention are not particularly limited as long as three-dimensional culture can be carried out. Examples of polysaccharides include hyaluronic acid, gellan gum, deacylated gellan gum (DAG), lambzan gum, daiyutan gum, xanthan gum, carrageenan, zantan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, etc. Examples include heparin sulfate, kerato sulfate, chondroitin sulfate, dermatan sulfate, ramnan sulfate, alginic acid and salts thereof. Examples of processed polysaccharides include, but are not limited to, chitin nanofibers, chitosan nanofibers, and cellulose nanofibers. As a method for preparing nanofibers from a polysaccharide such as chitin, a method known per se (see, for example, WO 2915/111686) may be used. Moreover, a commercially available product can also be used.

When three-dimensional culture is carried out using a polysaccharide or the like, the concentration of the polysaccharide or the like in the medium is not particularly limited as long as the three-dimensional culture can be carried out. The concentrations of specific polysaccharides and the like in the medium are illustrated below.

When deacylated gellan gum is used, it is usually 0.001% to 1.0% (weight / volume), for example 0.005% to 1.0% (weight / volume), preferably 0.003% to 0. 5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), even more preferably 0.01 to 0.05% (weight / volume), most preferably 0.01 It can be added to the medium in an amount of% to 0.02% (weight / volume). From the viewpoint of expressing the floating action, the lower limit of the deacylated gellan gum concentration in the medium is preferably 0.005% (weight / volume) or more, or 0.01% or more. From the viewpoint of enabling floating static culture, the lower limit of the deacylated gellan gum concentration in the medium is preferably 0.01% (weight / volume) or more. From the viewpoint of not substantially increasing the viscosity of the medium, the upper limit of the deacylated gellan gum concentration in the medium is 0.05% (weight / volume) or less. From the viewpoint of not substantially increasing the viscosity of the medium, the upper limit of the deacylated gellan gum may be 0.04 (weight / volume)% or less.

When carrageenan is used, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.01% to 0.1. % (Weight / volume), most preferably 0.02% to 0.1% (weight / volume). From the viewpoint of expressing the floating action and enabling floating static culture, the lower limit of the carrageenan concentration in the medium is preferably 0.01% or more. The upper limit of the carrageenan concentration in the medium is preferably 0.1% (weight / volume) or less. From the viewpoint of not substantially increasing the viscosity of the medium, the upper limit of carrageenan may be 0.04% (weight / volume) or less.

When cellulose nanofibers are used, they are usually 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0. In the medium to 5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), even more preferably 0.01% to 0.05% (weight / volume). Can be added.

When chitin nanofibers are used, they are usually 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0. In the medium to 5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), most preferably 0.03% to 0.07% (weight / volume). Can be added. From the viewpoint of expressing the floating action, the lower limit of the chitin nanofiber concentration in the medium is preferably 0.0001% (weight / volume) or more, 0.0003% (weight / volume) or more, and 0.0005% (weight / volume). Capacity) or more, or 0.001% (weight / capacity) or more. From the viewpoint of enabling floating static culture, the lower limit of chitin nanofibers in the medium is preferably 0.03% (weight / volume) or more. The upper limit of the chitin nanofiber concentration in the medium is preferably 0.1% (weight / volume) or less.

Water-insoluble nanofibers such as cellulose nanofibers and chitin nanofibers do not substantially increase the viscosity of the medium composition if the concentration is usually 0.1% (weight / volume) or less. That is, the medium containing such concentrations of nanofibers can be a liquid medium at 37 ° C.

It is also possible to use a combination of two or more kinds of polysaccharides and the like. The concentration of the polysaccharide or the like may be appropriately set within a range in which the cells can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid medium.

In the method of the present invention, the culture vessel used for three-dimensional culture includes a petri dish, a flask, a plastic bag, a Teflon (registered trademark) bag, a dish, a petride dish, a tissue culture dish, and a multi-dish that are generally used for cell culture. , Microplates, microwell plates, multiplates, multiwell plates, chamber slides, tubes, trays, culture bags, roller bottles and other culture vessels can be used. It is desirable that these culture vessels have low cell adhesion so that the (adhesive) cells to be cultured do not adhere to the culture vessels. As the cell non-adhesive culture container, the surface of the culture container is not artificially treated (for example, coating treatment with extracellular matrix or the like) for the purpose of improving the adhesion to cells, or the culture container. Those whose surface has been artificially treated for the purpose of reducing adhesion to cells can be used. Examples of such containers include Smilon Celtite Plate (manufactured by Sumitomo Bakelite Co., Ltd.), Prime Surface (registered trademark) plate (manufactured by Sumitomo Bakelite Co., Ltd.), ultra-low adhesive surface plate (manufactured by Corning Inc.), and Nuncron Sfera plate. (Manufactured by Thermo Fisher Scientific), etc., but is not limited to this.

The cell type capable of promoting proliferation by the method of the present invention is not particularly limited, but is derived from germ cells such as sperm and eggs, somatic cells constituting the living body, normal cell lines, cancer cell lines, precursor cells, stem cells, and living bodies. Examples thereof include cell types such as cells that have been isolated and artificially genetically modified, and cells that have been isolated from the living body and artificially exchanged nuclei. The origin of these cells is also not particularly limited, but rats, mice, rabbits, guinea pigs, squirrel monkeys, hamsters, voles, platypus, dolphins, whales, dogs, cats, goats, cows, horses, sheep, pigs, elephants, and commons. Cells derived from mammals such as marmosets, squirrel monkeys, voles, chimpanzees and humans are preferred. The tissue or organ from which the cells are derived is also not particularly limited as long as the desired effect of the present invention can be obtained, but the tissues include skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, and stomach. , Colon, small intestine, large intestine, bladder, prostate, testis, thoracic gland, muscle, connective tissue, bone, cartilage, vascular tissue, blood, heart, eye, brain or nerve tissue. Further, examples of the organ include, but are not limited to, organs such as liver, lung, kidney, heart, pancreas, stomach, spleen, small intestine, large intestine, and genital organ.

Normal cell lines include C3H10T1 / 2 (mouse embryonic fibroblasts), HEK293 (human fetal kidney cells), MDBK (bovine kidney-derived cells), MDCK (canine kidney tubule epithelial cells), and CHO-K1 (Chinese hamster ovary). Derivative cells), Vero cells (African green monkey kidney epithelial-derived cells), NIH3T3 (mouse fetal fibroblasts), HepaRG (hepatic cells, registered trademark), HUVEC (human umbilical vein endothelial cells), human primary cultured hepatocytes, etc. Be done. Of these, MDCK, HUVEC, CHO-K1 and Vero cells are particularly preferred. In addition, examples of cancer cell lines are not limited to the following, but human breast cancer cell lines include HBC-4, BSY-1, BSY-2, MCF-7, MCF-7 / ADR RES, and HS578T. , MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D, HeLa as a human cervical cancer cell line, A549, EKVX, HOP-62, HOP-92 as a human lung cancer cell line, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, DMS273, DMS114, human colon cancer cell lines Caco-2, COLO-205, HCC-2998, HCT-15, HCT-116 , HT-29, KM-12, SW-620, WiDr, DU-145, PC-3, LNCaP as human prostate cancer cell lines, U251, SF-295, SF-539 as human central nervous system cancer cell lines. , SF-268, SNB-75, SNB-78, SNB-19, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SKOV3, IGROV-1, human kidney cancer as human ovmortalized cell lines RXF-631L, ACHN, UO-31, SN-12C, A498, CAKI-1, RXF-393L, 786-0, TK-10 as cell lines, MKN45, MKN28, St-4, MKN- as human gastric cancer cell lines 1, MKN-7, MKN-74, LOX-IMVI, LOX, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC- as skin cancer cell lines Examples of 257, M14 and immortalized cell lines include CCRF-CRM, K562, MALT-4, HL-60TBRPMI8226, SR, UT7 / TPO, and Jurkat. Among these, human ovarian cancer cell line SKOV3, human cervical cancer cell line HeLa, human malignant melanoma-derived cell line A375, human epithelial-like cell cancer-derived cell line A431, and human gastric adenocarcinoma-derived cell line AGS , Human prostate cancer-derived cell line LNCap clone FGC, human colon adenocarcinoma-derived cell line HCT116, human alveolar basal epithelial adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell DU145 are particularly preferred. Further, a stem cell is a cell having both the ability to replicate itself and the ability to differentiate into cells of a plurality of other lines, and examples thereof are, but are not limited to, embryonic stem cells (ES). Cells), embryonic tumor cells, embryonic germ stem cells, artificial pluripotent stem cells (iPS cells), nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, Examples include stem cells and hair follicle stem cells. Examples of pluripotent stem cells include ES cells, embryonic reproductive stem cells, and iPS cells among the stem cells. Progenitor cells are cells in the process of differentiating from the stem cells into specific somatic cells or germ cells. As the stem cells, mesenchymal stem cells (MSCs) are particularly preferable.

The cell culture conditions (for example, temperature, carbon dioxide concentration, culture period, etc.) in the method of the present invention may be those known per se, or may be appropriately modified depending on the intended purpose. For example, the temperature at which cells are cultured is usually 25 to 39 ° C, preferably 33 to 39 ° C (eg, 37 ° C) for animal cells. The carbon dioxide concentration is usually 4 to 10% by volume in the culture atmosphere, preferably 4 to 6% by volume. The culturing period is usually 1 to 35 days, but it may be appropriately set according to the purpose of culturing.

In the method of the present invention, cells are cultured by 3D culture and their proliferation is promoted. Here, under three-dimensional culture, cells proliferate while forming spheres. Therefore, the method of the present invention can be paraphrased as "a method for producing a sphere".

2. Agent for adding 3D cell culture medium The present invention is also referred to as an agent for adding 3D cell culture medium (hereinafter, referred to as "agent of the present invention") containing the compound represented by the general formula (I) or a salt thereof. There is.)

The compounds compounded in the agent of the present invention, cell types capable of promoting proliferation, etc. are the same as those described in "1. Method for promoting cell proliferation".

In one aspect of the agents of the invention, the agents of the invention can be used to promote cell proliferation and / or sphere formation.

The agent of the present invention may contain one or more compounds represented by the general formula (I).

In addition, the agent of the present invention may contain other components other than the compound used in the present invention. Such components include, for example, water, physiological saline, dimethyl sulfoxide (DMSO), glycerin, propylene glycol, butylene glycol, and methanol, ethanol, butanol, propanol, but not particularly limited as long as the desired effects of the present invention can be obtained. Various alcohols, etc., etc. may be included. Moreover, the agent of this invention may be sterilized if necessary. The sterilization method is not particularly limited, and examples thereof include radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, and filter sterilization. The material of the filter part when performing filter sterilization (hereinafter, also referred to as filtration sterilization) is not particularly limited, but for example, glass fiber, nylon, PES (polyether sulfone), hydrophilic PVDF (polyvinylidene fluoride), cellulose. Examples thereof include mixed esters, cellulose acetate and polytetrafluoroethylene. The size of the pores of the filter is not particularly limited, but is preferably 0.1 to 10 μm, more preferably 0.1 to 1 μm, and most preferably 0.1 to 0.5 μm. In these sterilization treatments, the agent of the present invention may be in the form of a solid or a solution.

The compounding amount of the compound in the agent of the present invention is particularly high as long as the compounding amount is such that when the agent of the present invention is added to the three-dimensional cell culture medium, the medium has a concentration capable of exerting the desired effect of the present invention. Not limited. The lower limit of the concentration of the compound in the medium capable of exerting the desired effect of the present invention is usually 0.001 μM or more, preferably 0.01 μM or more, more preferably 0.1 μM or more, still more preferably 0.2 μM. As mentioned above, it may be 0.3 μM or more particularly preferably. The upper limit of the concentration is usually 100 μM or less, preferably 50 μM or less, more preferably 15 μM or less, still more preferably 5 μM or less, and particularly preferably 3 μM or less. In one embodiment, the final concentration of the compound in the medium is 0.001-100 μM, preferably 0.01-50 μM, more preferably 0.1-15 μM, even more preferably 0.2-5 μM, particularly preferably 0.3. It can be ~ 3 μM.

The agent of the present invention may have any shape at the time of delivery or storage. The agents of the present invention bind to formulated solids such as tablets, pills, capsules, granules, liquids such as solutions or suspensions dissolved in suitable solvents and solubilizers, or substrates and carriers. It can be in a suspended state. Additives for formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose and mannitt; and lubricants such as magnesium stearate and talc; polyvinyl Binders such as alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; plasticizers such as glycerin and the like can be mentioned. These additives are not limited to those mentioned above, and can be freely selected as long as they are available to those skilled in the art.

In the present specification, the term "agent of the present invention" can be replaced with the term "composition of the present invention" or "agent for adding a medium of the present invention".

The 3.3-dimensional cell culture medium present invention have the general formula (I) compound represented by (or, agents of the present invention) and a polysaccharide or its workpiece, 3-dimensional cell culture medium (hereinafter, "the present invention May be referred to as "medium").

The medium of the present invention is a medium that can be used for cell culture (for example, a commercially available medium listed in "1. Method for Promoting Cell Proliferation"), a compound represented by the general formula (I), and 3 cells. Contains polysaccharides or processed products thereof that enable dimensional culture. As described above, the polysaccharide or its processed product disperses in the medium to form a three-dimensional network. By using the network as a scaffold, three-dimensional culture (or suspension culture) of cells becomes possible.

In one aspect, the medium of the invention is a liquid medium.

The medium, compound, polysaccharide or processed product thereof constituting the medium of the present invention, their concentrations and the like are the same as those described in "1. Method for promoting cell proliferation".

The usefulness of the present invention will be specifically described in the following test examples, but the present invention is not limited to these examples. In the following examples, the concentration (%) of CO _{2 in the CO 2} incubator is shown by the volume% _{of CO 2 in the atmosphere.} In addition, PBS means phosphate buffered saline (manufactured by Sigma-Aldrich Japan), and FBS means fetal bovine serum (manufactured by Biological Industries). Further, (w / v) represents the weight per volume.

[Example 1] Action of cell proliferation agent on SKN cells According to the method of Patent Document 1 (WO2014 / 017513), 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, Sansho Co., Ltd.) A composition of Ham's F-12 medium (manufactured by Wako Junyaku Co., Ltd.) containing 10% (v / v) FBS was prepared. Subsequently, human uterine leiomyosarcoma cell line SKN cells (manufactured by JCRB cell bank) were suspended in the above-mentioned medium composition supplemented with deacylated gellan gum, and then 96-well flat-bottomed ultra-low adhesion surface microplates (manufactured by Corning Co., Ltd.). The wells of # 3474) were dispensed to 1000 cells / 135 μL / well per well (3D culture (3D)). Each plate was _{cultured in a CO 2} incubator (37 ° C., 5% CO ₂ ) in a stationary state. On the first day of culturing, 15 μL of each of five compounds (compounds A to E) dissolved in dimethyl sulfoxide (DMSO) was added to the medium so as to have a final concentration of 0, 1, 5, and 10 μM, and the culture was continued. Was continued for 4 days. 150 μL of ATP reagent (CellTiter-Glo® Luminescence Cell Viability Assay, manufactured by Promega) was added to the culture medium on the 5th day, suspended, and allowed to stand at room temperature for 10 minutes, and then FlexStation3 (Molecular Devices). The luminescence intensity (RLU value) was measured, and the number of living cells was measured by subtracting the luminescence value of the medium only.

Table 1 shows the relative values at the time of adding each compound when the RLU value (ATP measurement, emission intensity) without the compound added was set to 100%.

As shown in Table 1, the addition of compounds A to E markedly promoted cell proliferation of SKN cells in 3D culture. In addition, SKN cells under three-dimensional culture formed spheres in the medium.

[Example 2] Comparison of action of cell growth promoter on human mesenchymal stem cells under 3D culture Human bone marrow-derived mesenchymal stem cells (hBM-MSC, manufactured by Takara Bio Co., Ltd.) were used as a mesenchymal stem cell growth medium. Pre-cultured by a monolayer culture method (two-dimensional culture method (2D)) using 2 (manufactured by Takara Bio Co., Ltd.). In the 3D culture method, hBM-MSC is suspended in a medium composition supplemented with deacylated gellan gum, and then 2000 cells / 150 μL / well is obtained on a 96-well flat-bottom ultra-low adhesive surface plate (Corning, # 3474). Was sown. On the first day of culture, 15 μL of each compound dissolved in dimethyl sulfoxide (DMSO) was added to a final concentration of 0, 0.1, 0.5, 1, or 3 μM, and the culture was continued for 6 days. did. 135 μL of ATP reagent (CellTiter-Glo® Luminescence Cell Viability Assay, manufactured by Promega) was added to the culture medium on the 7th day, suspended, and allowed to stand at room temperature for 10 minutes, and then FlexStation3 (Molecular Devices). The luminescence intensity (RLU value) was measured, and the number of living cells was measured by subtracting the luminescence value of the medium only.

Table 2 shows the relative values at each compound addition concentration when the RLU value (ATP measurement, luminescence intensity) under the culture conditions to which no compound was added was set to 100%. In addition, "-" in the table means not implemented.

As shown in Table 2, compounds A to E increased the number of cells in hMSC under 3D culture. The hMSC cultured under the three-dimensional culture formed a sphere.

[Example 3] Effect of cell proliferation promoter on various human cancer cells and normal cells under three-dimensional culture Various human-derived cancer cells are pre-cultured (monolayer culture) in each medium as follows. It was. Human epithelial cell cancer-derived cell line A431 (ATCC, 10% FBS and 1% MEM non-essential amino acid solution (MEM Non-Essential Amino Acids solution; NEAA, Wako Pure Chemical Industries, Ltd.) containing Eagles's Minimum Essential Medium; EMEM (manufactured by Wako Pure Chemical Industries, Ltd.), human breast cancer-derived cell line MDA-MB-231 (manufactured by ATCC, 10% fetal bovine serum (FBS, manufactured by Corning)) Dulvecco's Modified Eagle's Medium; DMEM (manufactured by Wako Pure Chemical Industries, Ltd.)), human umbilical vein endothelial cell HUVEC (manufactured by Takara Bio, endothelial cell proliferation medium (manufactured by Takara Bio)). After washing these cells in the logarithmic growth phase with PBS, the adherent cells were added with a 0.25 w / v% trypsin-1 mmоl / L EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.) at 37 ° C. After incubating for 1 to 5 minutes and exfoliating, each medium was added, centrifuged, resuspended in the same medium, and each was collected as a single cell.

The above various cells were suspended in each medium containing or not containing deacylated gellan gum, and at a cell concentration of 700 to 2000 cells / 90 μL / well, a 96-well low-adhesion U-bottom plate (manufactured by Corning, deacylated gellan gum-free). (Medium-containing) or low-adhesion flat-bottomed plate (manufactured by Corning, deacylated gellan gum-containing medium (deacylated gellan gum concentration: 0.015 w / v%)) was inoculated (all under 3D culture conditions) at 37 ° C. After culturing in a 5% CO ₂ incubator overnight, a compound D solution dissolved in each medium so as to have a final concentration of 5 μM for MDA-MB-231, a final concentration of 10 μM for A431, and a final concentration of 5 μM for HUVEC was added. was added at 10 [mu] L / well, comparing the subjects were added to DMSO solution in the medium (DMSO final concentration 0.1%). continuing 37 ° C., 4 days after culturing at 5% CO ₂ incubator, 5 days 100 μL of ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, manufactured by Promega) was added to the culture medium of the above, suspended, allowed to stand at room temperature for 10 minutes, and then placed in FlexStation 3 (manufactured by Molecular Devices). The luminescence intensity (RLU value) was measured, and the number of living cells was measured by subtracting the luminescence value of the medium only.

When the RLU value (ATP measurement, emission intensity) without compound is 100%, those showing a value of 100% or more and less than 120% are Δ, and those showing a value of 120% or more and less than 150% are ○, 150. Table 3 shows a value of% or more as ⊚.

As shown in Table 3, the compounds of the present invention represented by Compound D promoted the growth of cancer cell lines and normal cells in 3D culture. The cancer cell line formed a sphere regardless of whether the low-adhesion U-bottom plate or the low-adhesion flat-bottom plate was used.

According to the present invention, the proliferation of cells under 3D culture can be easily promoted. Further, according to the present invention, spheres can be efficiently formed. Therefore, the present invention can efficiently produce high quality cells. Therefore, the present invention is very useful, for example, in the fields of regenerative medicine and drug discovery.

Claims (15)

A method for promoting cell proliferation, which comprises three-dimensionally culturing cells in a medium supplemented with the compound represented by the following general formula (I) or a salt thereof.

{In the formula, R ₁ is hydrogen, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an aryl group which may have a substituent, and R ₂ is hydrogen or an aryl group which may have a substituent. It is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or R ₁ and R ₂ are together- (CH ₂ ) _n- (in the formula, n is 2, 3, 4 or 5) may be formed, where R ₃ is hydrogen, -CONH-CH ₃ or -NHCO-CH ₃ }. The method of claim 1, wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines, and stem cells. The method of claim 2, wherein the normal cell line is selected from the group consisting of human umbilical vein endothelial cells (HUVEC), mouse embryonic fibroblasts (C3H10T1 / 2), and human fetal kidney cells (HEK293). The cancer cell lines are SKOV3 cells, HeLa cells, human malignant melanoma-derived cell line A375, human epithelial-like cell cancer-derived cell line A431, human gastric adenocarcinoma-derived cell line AGS, and human prostate cancer-derived cell line LNCap clone. The second or more selected from the group consisting of FGC, human colon adenocarcinoma-derived cell line HCT116, human alveolar basal epithelial adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell DU145. Method. The method according to claim 2, wherein the stem cell is a human mesenchymal stem cell (MSC). The method according to any one of claims 1 to 5, wherein the compound is the compound shown below or a salt thereof.

The method according to any one of claims 1 to 6, wherein the concentration of the compound in the medium is 0.001 to 100 μM. A three-dimensional cell culture medium addition agent containing the compound represented by the following general formula (I) or a salt thereof:

{In the formula, R ₁ is hydrogen, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an aryl group which may have a substituent, and R ₂ is hydrogen or an aryl group which may have a substituent. It is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or R ₁ and R ₂ are together- (CH ₂ ) _n- (in the formula, n is 2, 3, 4 or 5) may be formed, where R ₃ is hydrogen, -CONH-CH ₃ or -NHCO-CH ₃ }. The agent according to claim 8, wherein the cells are selected from the group consisting of normal cells, cancer cells, and stem cells. The agent according to claim 9, wherein the normal cell line is selected from the group consisting of human umbilical vein endothelial cells (HUVEC), mouse embryonic fibroblasts (C3H10T1 / 2), and human fetal kidney cells (HEK293). The cancer cells are SKOV3 cells, HeLa cells, human malignant melanoma-derived cell line A375, human epithelial-like cell cancer-derived cell line A431, human gastric adenocarcinoma-derived cell line AGS, and human prostate cancer-derived cell line LNCap clone FGC. The agent according to claim 9, which is selected from the group consisting of human colon adenocarcinoma-derived cell line HCT116, human alveolar basal epithelial adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell DU145. The agent according to claim 9, wherein the stem cell is a human mesenchymal stem cell (MSC). The agent according to any one of claims 8 to 12, wherein the compound is the compound shown below or a salt thereof.

A cell culture medium containing the agent according to any one of claims 8 to 13 and a polysaccharide or a processed product thereof. A method for producing a sphere, which comprises three-dimensionally culturing cells in a medium supplemented with the compound represented by the following general formula (I) or a salt thereof.

{In the formula, R ₁ is hydrogen, an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an aryl group which may have a substituent, and R ₂ is hydrogen or an aryl group which may have a substituent. It is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or R ₁ and R ₂ are together- (CH ₂ ) _n- (in the formula, n is 2, 3, 4 or 5) may be formed, where R ₃ is hydrogen, -CONH-CH ₃ or -NHCO-CH ₃ }.