WO2019117262A1 - Composition pour promouvoir l'adhésion cellulaire et méthode de culture cellulaire l'utilisant - Google Patents

Composition pour promouvoir l'adhésion cellulaire et méthode de culture cellulaire l'utilisant Download PDF

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WO2019117262A1
WO2019117262A1 PCT/JP2018/045960 JP2018045960W WO2019117262A1 WO 2019117262 A1 WO2019117262 A1 WO 2019117262A1 JP 2018045960 W JP2018045960 W JP 2018045960W WO 2019117262 A1 WO2019117262 A1 WO 2019117262A1
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cells
medium
stem cells
composition according
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泰斗 西野
昌史 岩上
脩成 岩本
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日産化学株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/72—Hydrazones
    • C07C251/86—Hydrazones having doubly-bound carbon atoms of hydrazone groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
    • C07C281/08—Compounds containing any of the groups, e.g. semicarbazides the other nitrogen atom being further doubly-bound to a carbon atom, e.g. semicarbazones
    • C07C281/14—Compounds containing any of the groups, e.g. semicarbazides the other nitrogen atom being further doubly-bound to a carbon atom, e.g. semicarbazones the carbon atom being further bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a composition and the like for promoting cell adhesion of adherent cells, and in particular, to a composition for promoting cell adhesion and a method for culturing cells characterized by using the composition. .
  • animal-derived cells are roughly divided into floating cells and adherent cells. Suspended cells are cells that do not require a scaffold for growth and proliferation, and adherent cells are cells that require a scaffold for growth and proliferation, but most of the cells that make up the organism have the latter adhesiveness. It is a cell.
  • adherent cells are cells that require a scaffold for growth and proliferation, but most of the cells that make up the organism have the latter adhesiveness. It is a cell.
  • problems such as low cell adhesion ratio or long time to cell adhesion may occur.
  • Non-patent Document 1 a method for promoting adhesion of cells to a culture vessel is required, and, for example, a method for promoting cell adhesion using an extracellular matrix has been reported.
  • Patent Documents 1 and 2 methods for promoting cell adhesion using chemically synthesized low molecular weight compounds and polymers have been reported.
  • An object of the present invention is to provide a composition for adding a medium capable of promoting adhesion of adherent cells to a culture vessel.
  • the present inventors have found that by adding a specific compound to a culture medium, adhesion of adherent cells to a culture vessel can be promoted extremely well,
  • the present invention has been completed by conducting further research based on it. That is, the present invention is as follows.
  • composition for promoting cell adhesion comprising a compound represented by the following general formula (I), or a salt thereof:
  • X is a single bond, -CH 2 COO-, -CONH-, or -NHCO-
  • R 1 is an optionally substituted alkyl group having 1 to 10 carbon atoms, substituted An aryl group which may have a group or —Y—NH—Z—Ar, wherein Y and Z each have a single bond or a carbon number of 1 to 6 which may have a substituent
  • Ar is an aryl group which may have a substituent
  • R 2 is an alkyl group having 1 to 6 carbon atoms which may have a substituent
  • R 3 Is a hydrogen atom or a hydroxyl group.
  • composition according to any one of [1] to [8], wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
  • the composition according to [9], wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
  • the composition according to [9], wherein the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
  • the composition of [9], wherein the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
  • hMSCs human mesenchymal stem cells
  • hiPSCs human induced pluripotent stem cells
  • the medium according to [13], wherein the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
  • the medium according to [14], wherein the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
  • the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
  • the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
  • a method for promoting adhesion of cells to a culture vessel comprising adding the composition according to any one of [1] to [8] to a culture medium.
  • the cells are selected from the group consisting of normal cell lines, cancer cell lines and stem cells.
  • the normal cell line is HUVEC, C3H10T1 / 2 or HEK293.
  • the cancer cell line is HeLa cells, A431, HCT116, or MCF7.
  • the stem cells are human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs).
  • the present invention it is possible to promote the adhesion of adherent cells to a culture vessel, in which adhesion to a substrate or the like is essential in cell growth. This makes it possible to adhere the cells efficiently, and to carry out studies using the cells more rapidly and efficiently than in the past.
  • the adherent cells cultured using the present invention are stem cells (eg, induced pluripotent stem cells)
  • the present invention promotes adhesion of the stem cells to a culture vessel while the cells are being cultured. An undifferentiated state of stem cells can be suitably maintained.
  • FIG. 1 shows that when the composition of the present invention is added to the culture medium at stepwise concentrations, cell adhesion to the culture vessel of the HEK 293 cell line is promoted in a concentration dependent manner.
  • halogen atom is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
  • halogeno group is fluoro, chloro, bromo or iodo.
  • alkyl group means a linear or branched alkyl group, and specifically, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- Groups such as pentyl, isopentyl, tert-pentyl, neopentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like can be mentioned.
  • Aryl includes 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, examples include phenyl, indenyl, naphthyl, fluorenyl and the like. In particular, the aryl group may be C 6-10 aromatic phenyl, indenyl, naphthyl.
  • alkylene group means a linear or branched alkylene group, and specifically, methylene, ethylene, n-propylene, isopropylene, propylidene, n-butylene, sec-butylene, tert-butylene, n -Groups such as pentylene, sec-pentylene, tert-pentylene, n-hexylene, sec-hexylene, tert-hexylene and the like can be mentioned.
  • alkyl group may have a substituent, and examples of such a substituent include the following. (1) halogeno group, (2) hydroxy group, (3) cyano group, (4) nitro group, (5) Carboxyl group, (6) Alkenyl group (C 2-10 alkenyl group; for example, vinyl, allyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, butadienyl, hexatrienyl, and each isomer thereof), (7) alkynyl group (C 2-10 alkynyl group; for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl and each isomer thereof), (8) a halogenoalkyl group (eg, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl,
  • carbamoyl group (27) carbamoyl group, (28) a carbamoyl group mono- or di-substituted with an alkyl group (as defined for the “alkyl group” in the above (26)) (eg, methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, ethylmethylcarbamoyl) (29) sulfamoyl group, (30) A sulfamoyl group mono- or di-substituted with an alkyl group (as defined for the "alkyl group” in the above (26)) (eg, methylsulfamoyl, ethylsulfamoyl, dimethylsulfamoyl, diethylsulfamoyl, Ethyl methyl sulfamoyl), (31) an alkanoyl group (eg, a
  • the “C 1-6 alkyl group” is one having 1 to 6 carbon atoms among the above “alkyl groups”, and the “C 6-10 aryl group” is one of the above “aryl groups”. Among them, those having 6 to 10 carbon atoms.
  • Specific examples of the acyl group include acetyl group, propionyl group, butyroyl group, isobutyroyl group, valeroyl group, isovaleroyl group, pivaloyl group, hexanoyl group, acryloyl group, methacryloyl group, crotonoyl group, isocrotonoyl group, benzoyl group and naphthoyl group.
  • an alkoxycarbonylamino group eg, a carbonylamino group substituted with an alkoxy group (as defined in the above (12)
  • an alkylsulfonyl group eg, a sulfonyl group substituted with an alkyl group (as defined as the “alkyl group” in the above (26)
  • an alkylsulfinyl group eg, a sulfinyl group substituted with an alkyl group (as defined as the “alkyl group” in the above (26)
  • (40) alkoxycarbonyl group eg, methoxycarbonyl group, ethoxycarbonyl group
  • they may be the same or different.
  • the compounds of formula (I) may be in the form of salts.
  • the salt of the compound represented by the formula (I) include salts with inorganic acids such as hydrochloric acid and hydrobromic acid, as well as acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, And salts with organic acids such as succinic acid, citric acid and benzoic acid.
  • the reaction mixture can be precipitated by adding distilled water, or when not precipitated, it can be subjected to a usual post treatment such as extraction after extraction with an organic solvent to obtain the desired compound of the present invention.
  • a usual post treatment such as extraction after extraction with an organic solvent to obtain the desired compound of the present invention.
  • it can be separated and purified by any purification method such as recrystallization, column chromatograph, thin layer chromatograph, liquid chromatograph or the like.
  • reaction mixture After completion of the reaction, the reaction mixture can be subjected to usual post treatments such as extraction with solvent and concentration after addition of distilled water to obtain the desired compound of the present invention.
  • it can be separated and purified by any purification method such as recrystallization, column chromatograph, thin layer chromatograph, liquid chromatograph or the like.
  • composition for promoting cell adhesion provides a composition for promoting cell adhesion (hereinafter sometimes referred to as "the composition of the present invention"), which contains the following compound.
  • the composition of the present invention can promote cell adhesion of cells (particularly adherent cells) to a culture vessel by adding to a culture medium for cell culture.
  • the compounds included in the composition of the present invention have the general formula (I):
  • X is a single bond, -CH 2 COO-, -CONH-, or -NHCO-
  • R 1 is an optionally substituted alkyl group having 1 to 10 carbon atoms, substituted An aryl group which may have a group or —Y—NH—Z—Ar, wherein Y and Z each have a single bond or a carbon number of 1 to 6 which may have a substituent
  • Ar is an aryl group which may have a substituent
  • R 2 is an alkyl group having 1 to 6 carbon atoms which may have a substituent
  • R 3 Is a hydrogen atom or a hydroxyl group.
  • R 1 is an alkyl group having 1 to 10 carbon atoms which may have a substituent (more preferably a substituent
  • R 2 is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably substituted).
  • R 1 is an alkyl group having 1 to 6 carbon atoms, particularly preferably an ethyl group
  • R 3 is a hydrogen atom.
  • R 1 is an alkyl group having 1 to 10 carbon atoms which may have a substituent (preferably, the number of carbons which may have a substituent)
  • R 2 is an alkyl group of 1 to 6, particularly preferably an ethyl group
  • R 2 is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably has no substituent)
  • R 1 is an alkyl group having 1 to 6 carbon atoms, particularly preferably an ethyl group
  • R 3 is a hydrogen atom, or R 1 is a C 1 to 10 carbon atom which may have a substituent.
  • R 2 is an alkyl group (more preferably, an alkyl group having 1 to 6 carbon atoms having a substituent, particularly preferably a benzyl group), and R 2 is an optionally substituted carbon atom having 1 to 6 carbon atoms 6 alkyl groups (more preferably, alkyl groups having 1 to 6 carbon atoms which have no substituent, Particularly preferred is an ethyl group), and R 3 is a hydrogen atom.
  • R 1 is an aryl group which may have a substituent (more preferably, an aryl group having no substituent, particularly preferably a phenyl group)
  • R 2 is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably an alkyl group having 1 to 6 carbon atoms which does not have a substituent, particularly preferably an ethyl group)
  • R 3 is a hydrogen atom.
  • R 1 is -Y-NH-Z-Ar, where, when Y is a single bond, Z has a single bond or a substituent
  • Optionally substituted alkylene group (more preferably a single bond or an alkylene group having no substituent, particularly preferably a single bond or a methylene group)
  • Ar is a substituent (more preferably a halogen atom,
  • An aryl group optionally having a methyl group, a hydroxyl group or a methoxy group more preferably, an aryl group having a hydroxyl group or an aryl group having no substituent, particularly preferably a phenyl group or a phenyl group having a hydroxyl group
  • R 2 is an alkyl group having 1 to 6 carbon atoms which may have a substituent (more preferably, an alkyl group having 1 to 6 carbon atoms which does not have a substituent, particularly preferably Echi A group)
  • R 3 is hydrogen atom.
  • X is -NHCO- and Y is an alkylene group having 1 to 6 carbon atoms which may have a substituent (more preferably, an alkylene group having 1 to 6 carbon atoms which does not have a substituent) And particularly preferably a methylene group, an ethylidene group or a propylidene group), Z is a single bond, Ar is an aryl group which may have a substituent (more preferably, it has a substituent) Aryl group, particularly preferably a halogeno group, a methyl group, a phenyl group having a hydroxyl group or an ethoxy group, or a naphthyl group), and R 2 is an optionally substituted C 1 to C 6 carbon atom alkyl group (more preferably an alkyl group having 1 to 6 carbon atoms having no substituent, particularly preferably, a methyl group, an ethyl group or an isopropyl group) is, R 3 is a hydrogen
  • the compounding amount of the above-mentioned compound in the composition of the present invention is not particularly limited as long as the medium has a concentration at which the desired effect of the present invention can be exhibited when the composition of the present invention is added to the medium.
  • the lower limit of the concentration of the above-mentioned compound in the medium is usually 0.001 ⁇ M or more, preferably 0.01 ⁇ M or more, more preferably 0.1 ⁇ M or more, more preferably as a concentration at which the desired effect of the present invention can be exerted. May be 1 ⁇ M or more, particularly preferably 10 ⁇ M or more.
  • the upper limit value of the concentration may be usually 100 ⁇ M or less, preferably 50 ⁇ M or less, particularly preferably 20 ⁇ M or less, but is not limited thereto.
  • compositions of the present invention may be in any form as provided or stored.
  • the composition may be bound to a formulated solid such as a tablet, pill, capsule or granule, a suitable solvent and a liquid such as a solution or suspension dissolved with a solubilizer, or a substrate or carrier.
  • Can be Additives at the time of formulation include preservatives such as p-hydroxybenzoic acid esters; excipients such as lactose, glucose, sucrose, and mannitol; lubricants such as magnesium stearate and talc; polyvinyl Binders such as alcohol, hydroxypropyl cellulose and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. These additives are not limited to those described above, and can be freely selected by those skilled in the art if available.
  • Cell types suitable for use in the composition of the present invention may be cells of adherent cells.
  • Examples of such cells include somatic cells, normal cell lines, cancer cell lines, progenitor cells, stem cells, cells isolated from living bodies and artificially modified genetically, and artificial cells isolated from living bodies. And cells such as cells whose nuclei have been exchanged, but are not limited thereto.
  • the origin of these cells is also not particularly limited, but rat, mouse, rabbit, guinea pig, squirrel, hamster, vole, platypus, dolphin, whale, dog, cat, goat, cow, horse, sheep, pig, elephant, common Preferred are cells derived from mammals such as marmosets, squirrel monkeys, rhesus monkeys, chimpanzees and humans.
  • the tissue or organ from which the cells are derived is not particularly limited as long as the desired effect of the present invention can be obtained, and examples of the tissue include skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, Tissues such as stomach, colon, small intestine, large intestine, spleen, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, blood vessel tissue, blood, heart, eye, brain or nerve tissue can be mentioned. Further, examples of the organ include organs such as liver, lung, kidney, heart, pancreas, stomach, spleen, small intestine, large intestine, genital organ and the like.
  • examples of normal cell lines include C3H10T1 / 2 (mouse embryonic fibroblasts), HUVEC (human umbilical vein endothelial cells), HEK293 (human fetal kidney cells), MDBK (bovine kidney-derived cells), MDCK (MDCK) Canine renal tubular epithelial cells), Vero, NIH 3 T 3 (mouse fetal fibroblasts), HepaRG (hepatocytes, registered trademark), human primary culture hepatocytes and the like can be mentioned.
  • C3H10T1 / 2 mouse embryonic fibroblasts
  • HUVEC human umbilical vein endothelial cells
  • HEK293 human fetal kidney cells
  • MDBK bovine kidney-derived cells
  • MDCK MDCK
  • Vero Vero
  • NIH 3 T 3 mamouse fetal fibroblasts
  • HepaRG hepatocytes, registered trademark
  • human primary culture hepatocytes and the like can be mentioned.
  • cancer cell lines include, but are not limited to, human breast cancer cell lines such as HBC-4, BSY-1, BSY-2, MCF-7, MCF-7 / ADR RES, HS578T. , MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D, HeLa as a human cervical cancer cell line, A549 as a human lung cancer cell line, EKVX, HOP-62, HOP-92, 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-1 as a human prostate cancer cell line 5, PC-3, LNCaP, U251, SF-295, SF-539, SF-268, SNB-75, S
  • stem cell means a cell having both the ability to replicate itself and the ability to differentiate into cells of multiple other lineages, and examples thereof include, but are not limited to: But embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPSCs), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, reproductive Examples include stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells and the like. Pluripotent stem cells include ES cells, embryonic germ stem cells, and induced pluripotent stem cells among the stem cells.
  • ES cells embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • neural stem cells hematopoietic stem cells
  • mesenchymal stem cells mesenchymal stem cells
  • hepatic stem cells pancreatic stem cells
  • pancreatic stem cells pancreatic stem cells
  • muscle stem cells reproductive Examples include stem
  • the precursor cells are cells in the process of differentiating from the stem cells to specific somatic cells or germ cells.
  • stem cells human mesenchymal stem cells (hMSCs) or human induced pluripotent stem cells (hiPSCs) are particularly preferable.
  • composition of the present invention can be replaced with the term “agent of the present invention” or "cell adhesion promoter of the present invention”.
  • the present invention provides a culture medium for cell culture (hereinafter sometimes referred to as “the culture medium of the present invention”) containing the composition of the present invention.
  • the culture medium of the present invention By using the medium of the present invention, cell adhesion of cells (especially adherent cells) to a culture vessel can be promoted.
  • the concentration of the compound contained as an active ingredient in the culture medium of the present invention is not particularly limited as long as the desired effect of the present invention can be obtained, but the lower limit of the concentration is usually 0.001 ⁇ M or more, preferably 0.01 ⁇ M or more More preferably, it may be 0.1 ⁇ M or more, more preferably 1 ⁇ M or more, and particularly preferably 10 ⁇ M or more. Also, the upper limit value of the concentration may be usually 100 ⁇ M or less, preferably 50 ⁇ M or less, particularly preferably 20 ⁇ M or less.
  • the medium of the present invention can be the same as the composition of a known medium except that the composition of the present invention is blended.
  • the medium of the present invention can be prepared by adding the composition of the present invention to a commercially available medium.
  • the commercially available medium which can be used as the medium of the present invention by adding the composition of the present invention is not particularly limited as long as the desired effect can be obtained, for example, Dulbecco's Modified Eagle's Medium; DMEM), Ham's F12 medium (Ham's Nutrient Mixture F12), DMEM / F12 medium, McCoy's 5A medium (McCoy's 5A medium), Eagle MEM (Eagle's Minimum Essential Medium; EMEM), ⁇ MEM (alpha Modified Eagle's Minimum Essential Medium; ⁇ MEM) , MEM (Minimum Essential Medium), RPMI 1640 medium, chair F) Modified Dulbecco's Medium (IMDM), MCDB 131 Medium, William Medium E, IPL 41 Medium, Fischer's Medium, StemPro 34 (Invitrogen), X-VIVO 10 (Kenbrex), X-VIVO
  • 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 matrices, physiologically active substances and the like may be added according to the purpose.
  • the culture of cells in the medium of the present invention may be carried out using a petri dish, flask, plastic bag, Teflon bag, dish, petri dish, dish for tissue culture, multidish, microplate, microwell, which is generally used for cell culture. It can be carried out using culture vessels such as plates, multiplates, multiwell plates, chamber slides, tubes, trays, culture bags, roller bottles and the like. In one embodiment, the culture vessel may be artificially treated (for example, coated with an extracellular matrix or the like) in order to improve the adhesion to cells.
  • Examples of such containers are Matrigel (registered trademark), Geltrex (registered trademark), collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, vitronectin, tenascin, selectin, hyaluronic acid, Although the container which coated fibrin etc. is mentioned, it is not limited to this.
  • the cells capable of promoting cell adhesion by the medium of the present invention are the same as the cells described in “1. Composition for promoting cell adhesion”.
  • the present invention provides a method for promoting cell adhesion (hereinafter sometimes referred to as "the method of the invention"), which comprises adding the composition of the present invention to a culture medium. provide.
  • the method of the present invention can promote cell adhesion of cells (especially adherent cells) to a culture vessel.
  • the culture medium used in the method of the present invention is not particularly limited as long as the desired effect can be obtained.
  • cell culture conditions for example, temperature, carbon dioxide concentration, culture period, etc.
  • the temperature at which the cells are cultured is usually 25 ° C. to 39 ° C., preferably 33 ° C. to 39 ° C. (eg, 37 ° C.) for animal cells.
  • the carbon dioxide concentration is usually 4% by volume to 10% by volume, preferably 4% by volume to 6% by volume, in the culture atmosphere.
  • the culture period is usually 1 to 35 days, but may be appropriately set according to the purpose of culture.
  • the medium used in the method of the present invention may be the medium of the present invention.
  • the concentration of the compound serving as the active ingredient, the suitable cell type and the like in the method of the present invention are the same as those described in “1. Composition for promoting cell adhesion”.
  • the concentration (%) of CO 2 in the CO 2 incubator is shown by volume% of CO 2 in the atmosphere.
  • PBS phosphate buffered saline (manufactured by Sigma Aldrich Japan)
  • FBS fetal calf serum (manufactured by Biological Industries).
  • (w / v) represents weight per volume.
  • the intermediate compound (Compound No. 105) (5.11 g, 26.4 mmol) obtained as described above is dissolved in methanol (26 mL), hydrazine monohydrate (12.8 mL, 264 mmol) is added, and room temperature is obtained. The mixture was stirred for 4.5 hours. Water (150 mL) was added and extracted with methylene chloride (30 mL ⁇ 5). The organic layer is washed with saturated brine (100 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The solid obtained is suspended and washed with IPE, and H-7 (4.60 g, 23.8 mmol, Yield 90%) was obtained as a colorless solid.
  • Dissolve 110 (340 mg, 1.9 mmol) obtained as above in methanol (3.8 mL), add hydrazine monohydrate (0.18 mL, 3.8 mmol) and stir at 60 ° C. for 24 hours did. Hydrazine monohydrate (0.36 mL, 7.4 mmol) was added and stirred at 60 ° C. for additional 17 hours.
  • Dissolve 149 (200 mg, 1.6 mmol) obtained as described above in methylene chloride (2 mL) and water (2 mL), add sodium hydrogen carbonate (0.27 g, 3.2 mmol), and cool under ice cooling. Acid phenyl (136) (0.22 mL, 1.7 mmol) was slowly added dropwise. It stirred at room temperature for 20 hours, ethyl acetate (20 mL) and water (20 mL) were added, and it liquid-separated. The organic layer is washed with saturated brine (20 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure.
  • Example 1 Action on mesenchymal stem cells
  • Human bone marrow-derived mesenchymal stem cells (hMSC, manufactured by PromoCell) were precultured for 7 to 10 days using mesenchymal stem cell growth medium 2 (manufactured by PromoCell).
  • the resulting hMSCs were suspended in the same medium to 2.0 ⁇ 10 5 cells / mL.
  • k-1 H-1, k-1: H-7, k-1: H-10, k-1: I-1, k-1 dissolved in DMSO so as to give a final concentration of 10 ⁇ M:
  • a B-1 or k-1: J-1 solution was added, and 0.5 mL / well was seeded in a 24-well flat bottom plate (Corning # 35114).
  • DMSO was added to a final concentration of 0.1%. The plate was incubated at 37 ° C. in a 5% CO 2 incubator for 20 minutes.
  • the medium is washed three times with 500 ⁇ L of PBS to remove PBS, and then 0.25 w / v% trypsin-1 mmol / L EDTA (ethylenediaminetetraacetic acid) solution (Wako Pure Chemical Industries, Ltd.) in PBS
  • the cells were detached by adding 400 ⁇ L of a 5-fold diluted solution and incubating at 37 ° C. for 2 to 5 minutes. Further, the same volume of medium was added, transferred to another container, and centrifuged at 400 g for 4 minutes to remove the supernatant.
  • Example 2 Effects on Various Cell Lines Various human-derived cells were cultured using each of the following media.
  • Human cervical cancer-derived cell line HeLa (manufactured by American Type Culture Collection (ATCC), 10% fetal bovine serum (FBS, manufactured by Corning)) Dulbecco's Modified Eagle's Medium; DMEM (Wako Pure Chemical Industries, Ltd.) ), Eagle 'containing Eagle's cell line A431 derived from human epithelial-like cell carcinoma (manufactured by ATCC, 10% FBS and 1% MEM non-essential amino acid solution (NEAA, manufactured by Wako Pure Chemical Industries, Ltd.)) s Minimum Essential Medium; EMEM (Wako Pure Chemical Industries, Ltd.), human colon adenocarcinoma cell line HCT116 (DS Pharma Biomedical Co., Ltd., McCoy's 5A Me with 10% FBS) ium (Sigma-Aldrich), human breast cancer cell line MCF7 (ATCC, 10% FBS and 1%
  • trypsin-1 mmol / L EDTA ethylenediaminetetraacetic acid
  • trypan blue manufactured by Wako Pure Chemical Industries, Ltd.
  • K-1 H-1, k-1: H-7, k-1: H-10, k-2: H-7, k-2: H-1, k-1: I- dissolved in DMSO 1, k-1: B-1, k-1: D-1, or k-1: J-1 solution is added to each medium in which the various cells are suspended to a final concentration of 10 ⁇ M.
  • 8000 cells / 100 ⁇ L / well were seeded on a 96-well adhesive plate (Corning # 3585).
  • a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%).
  • the luminescence intensity was measured with EnSpire (manufactured by PerkinElmer), and the number of cells adhering to the plate was measured by subtracting the luminescence value of the medium alone.
  • the compound-free RLU value ATP measurement, luminescence intensity
  • those showing values of 100% or less are "-"
  • those showing values of 103% or more are " ⁇ ”
  • 120% or more are shown in Table 2
  • those having a value of “o” are shown in Table 2 as “o”.
  • the blanks indicate that the test has not been conducted.
  • composition of the present invention was shown to have a cell adhesion promoting effect on various cell lines.
  • Example 3 Effects of the composition of the present invention on normal cells Human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells: HUVEC) are used as a medium of Endothelial Cell Growth Medium (Ready-to-use) (PromoCell). Culture was performed. The above cells in logarithmic growth phase are washed with PBS, added with 0.25 w / v% trypsin-1 mmol / L EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.), and kept at 37 ° C. for 3 minutes. Incubate and detach, add media, centrifuge and remove supernatant.
  • HUVEC Human Umbilical Vein Endothelial Cells
  • trypsin-1 mmol / L EDTA ethylenediaminetetraacetic acid
  • the above cells were adjusted to a final concentration of 10 ⁇ M with a solution of k-1: I-1, k-1: B-1, k-1: D-1 or k-1: J-1 dissolved in DMSO.
  • the suspension was added to each culture medium, and 8000 cells / 100 ⁇ L / well were seeded on a 96-well adhesion plate (Corning # 3585).
  • a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%). After incubating this plate for 30 minutes in a 5% CO 2 incubator at 37 ° C., the culture solution was aspirated, and cells not adhered to the culture plate were removed by washing twice using each medium.
  • Example 4 Concentration dependent action on HEK293 cell line k-1: J-1 solution in DMSO is added to the culture medium to a final concentration of 0.1, 1 or 10 ⁇ M, and HEK293 cells are further added. After adding so as to be able to inoculate each culture medium at a cell concentration of 8000 cells / 100 ⁇ L / well, the cells were seeded on a 96-well adhesion plate (Corning # 3585). As a control, a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%). After stationary culture at 37 ° C.
  • k-1 J-1 promoted cell adhesion of HEK 293 in a concentration-dependent manner.
  • Example 5 Effect of the composition of the present invention on fibroblast cell line C3H10T1 / 2 Mouse embryonic fibroblast C3H10T1 / 2 (manufactured by DS Pharma Biomedical Co., Ltd.) is 10 (v / v)% FBS (manufactured by Corning) Culturing was carried out using BME medium (manufactured by Thermo Fisher Scientific Co.) containing L) -glutamine-penicillin-streptomycin stabilization solution (manufactured by Sigma-Aldrich).
  • the above cells in logarithmic growth phase are washed with PBS, added with 0.25 w / v% trypsin-1 mmol / L EDTA (ethylenediaminetetraacetic acid) solution (manufactured by Wako Pure Chemical Industries, Ltd.), and kept at 37 ° C. for 3 minutes. Incubate and detach, add media, centrifuge and remove supernatant. Thereafter, after resuspension in the same medium, a part was suspended in trypan blue (manufactured by Wako Pure Chemical Industries, Ltd.), and the number of living cells was counted by TC-20 (manufactured by BIO-RAD).
  • trypsin-1 mmol / L EDTA ethylenediaminetetraacetic acid
  • k-1 I-1, k-1: B-1 or k-1: J-1 solution dissolved in DMSO to each medium in which the above-mentioned cells were suspended to a final concentration of 10 ⁇ M
  • 8000 cells / 100 ⁇ L / well were seeded on a 96-well culture plate (Corning # 351172).
  • a cell suspension containing DMSO was seeded (final concentration of DMSO 0.1%).
  • the culture solution was aspirated and cells not adhered to the culture plate were removed by washing twice using the above-mentioned medium Thereafter, 100 ⁇ L / well of medium was added.
  • Example 6 Effects of the composition of the present invention on human induced pluripotent stem cells
  • Human induced pluripotent stem cells hiPSC, iPS manufactured by Academia Japan
  • 253G1 strain 253G1 strain
  • mTeSR (registered trademark) 1 medium manufactured by STEMCELL TECHNOLOGIES
  • the resulting hiPSCs were suspended in the same medium to 1.0 ⁇ 10 4 cells / mL.
  • a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 ⁇ M, and a 12-well flat bottom plate (Corning # 351143) is added to 1
  • the cells were seeded at 0 mL / well.
  • DMSO was added to a final concentration of 0.1%.
  • the plate was cultured at 37 ° C. in a 5% CO 2 incubator for 24 hours. After culture, remove the medium, add 500 ⁇ L of medium afresh, add and suspend the same amount of ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, Promega), and allow to stand for 10 minutes at room temperature.
  • CellTiter-Glo registered trademark
  • Luminescent Cell Viability Assay Promega
  • Example 7 The effect of the composition of the present invention on human induced pluripotent stem cells Human induced pluripotent stem cells (hiPSC, iPS manufactured by Academia Japan Co., Ltd.) 201B7 strain, 7 to 7 using StemFit medium (manufactured by Ajinomoto Co.) It was cultured for 14 days. The resulting hiPSCs were suspended in the same medium to 1.0 ⁇ 10 4 cells / mL.
  • hiPSC Human induced pluripotent stem cells
  • 201B7 strain 7 to 7 using StemFit medium (manufactured by Ajinomoto Co.) It was cultured for 14 days.
  • the resulting hiPSCs were suspended in the same medium to 1.0 ⁇ 10 4 cells / mL.
  • a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 ⁇ M, and a 12-well flat bottom plate (Corning # 351143) is added to 1
  • the cells were seeded at 0 mL / well.
  • DMSO was added to a final concentration of 0.1%.
  • the plate was cultured at 37 ° C. in a 5% CO 2 incubator for 24 hours. After culture, remove the medium, add 500 ⁇ L of medium afresh, add and suspend the same amount of ATP reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay, Promega), and allow to stand for 10 minutes at room temperature.
  • CellTiter-Glo registered trademark
  • Luminescent Cell Viability Assay Promega
  • Example 8 Evaluation of undifferentiated human artificial pluripotent stem cells when using the composition of the present invention
  • Human artificial pluripotent stem cells hiPSC, manufactured by iPS Academia Japan
  • StemFit medium manufactured by Ajinomoto Co.
  • the resulting hiPSCs were suspended in the same medium to 2.0 ⁇ 10 4 cells / mL.
  • a solution of k-1: B-1 or k-1: J-1 dissolved in DMSO is added to a final concentration of 10 ⁇ M, and a 6-well flat bottom plate (Corning # 351146) is added to 1 .5 ml / well was seeded.
  • DMSO was added to a final concentration of 0.1%.
  • the plate was cultured at 37 ° C. in a 5% CO 2 incubator for 120 hours. During the culture, medium exchange was performed using StemFit medium not containing the above-mentioned compound after 24, 72 hours. After culture, the medium is removed and 1 mL / well of PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.) is added and washed, then 1 mL / well of 0.5 mmol / L-EDTA / PBS solution (Nacalai Tesque, Inc.) ) was added and allowed to stand for 10 minutes in a 37 ° C., 5% CO 2 incubator.
  • PBS PBS
  • the detached cells were collected in a 15 mL centrifuge tube, and centrifuged at 200 g for 3 minutes (multiple tension thermostat EIX-136, manufactured by Tomy Seiko Co., Ltd.). The supernatant is then removed and 200 ⁇ L of TrypLE® Select (1 ⁇ ) (Gibco) was added, and left in a thermostat at 37 ° C. for 2 minutes. After 2 minutes, remove the 15 mL centrifuge tube from the thermostat, add 5 mL of 10% FBS-containing D-MEM (High Glucose) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and centrifuge at 200 g for 3 minutes. went.
  • FBS-containing D-MEM High Glucose
  • PE Mouse anti-Human TRA-1-60 Antigen and Alexa Fluor (registered trademark) 647 Mouse anti-SSEA-4 both manufactured by BD Bioscience
  • 5 ⁇ L of each was added and allowed to stand at room temperature for 30 minutes.
  • 5 ⁇ L each of PE Mouse IgM, I Isotype Control and Alexa Fluor (registered trademark) 647 Mouse IgG 3, Is Isotomype Control both manufactured by BD Bioscience) were added as Isotoype Control, and allowed to stand at room temperature for 30 minutes.
  • adhesion of adherent cells to a culture vessel can be promoted, whereby the culture of adherent cells can be made efficient.
  • the present invention is useful in the field of research and medicine using adherent cells.

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Abstract

La présente invention concerne une composition pour promouvoir l'adhésion cellulaire contenant un composé représenté par la formule générale (I) ou un sel de celui-ci. {Dans la formule, chaque symbole est tel que défini dans la description.}
PCT/JP2018/045960 2017-12-13 2018-12-13 Composition pour promouvoir l'adhésion cellulaire et méthode de culture cellulaire l'utilisant WO2019117262A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2020067477A1 (fr) * 2018-09-28 2020-04-02 日産化学株式会社 Additif pour milieu destiné à favoriser la production de facteur paracrine
WO2020158840A1 (fr) * 2019-01-30 2020-08-06 日産化学株式会社 Agent anticancéreux
WO2020158841A1 (fr) * 2019-01-30 2020-08-06 日産化学株式会社 Composé hydrazide et inhibiteur de kinase
WO2022085781A1 (fr) * 2020-10-23 2022-04-28 日産化学株式会社 Agent thérapeutique pour troubles hématopoïétiques

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WO1995022599A1 (fr) * 1994-02-18 1995-08-24 Teijin Limited Procede de mise en culture de cellules animales
JPH11322614A (ja) * 1998-05-15 1999-11-24 Hogi Medical:Kk 細胞接着促進効果を有する創傷止血材
WO2009154201A1 (fr) * 2008-06-18 2009-12-23 国立大学法人京都大学 Agent promoteur de l’adhésion cellulaire et procédé destiné à promouvoir l’adhésion cellulaire
JP2017517522A (ja) * 2014-05-30 2017-06-29 インスティテュート オブ ファーマコロジー アンド トキシコロジー アカデミー オブ ミリタリー メディカル サイエンシズ ピー．エル．エー．チャイナ 置換アセチドラジド誘導体、その調製方法及び使用
WO2019022222A1 (fr) * 2017-07-28 2019-01-31 日産化学株式会社 Composition additive pour milieu de culture, composé additif pour milieu de culture, et procédé de culture de cellules ou de tissu l'utilisant

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WO1995022599A1 (fr) * 1994-02-18 1995-08-24 Teijin Limited Procede de mise en culture de cellules animales
JPH11322614A (ja) * 1998-05-15 1999-11-24 Hogi Medical:Kk 細胞接着促進効果を有する創傷止血材
WO2009154201A1 (fr) * 2008-06-18 2009-12-23 国立大学法人京都大学 Agent promoteur de l’adhésion cellulaire et procédé destiné à promouvoir l’adhésion cellulaire
JP2017517522A (ja) * 2014-05-30 2017-06-29 インスティテュート オブ ファーマコロジー アンド トキシコロジー アカデミー オブ ミリタリー メディカル サイエンシズ ピー．エル．エー．チャイナ 置換アセチドラジド誘導体、その調製方法及び使用
WO2019022222A1 (fr) * 2017-07-28 2019-01-31 日産化学株式会社 Composition additive pour milieu de culture, composé additif pour milieu de culture, et procédé de culture de cellules ou de tissu l'utilisant

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020067477A1 (fr) * 2018-09-28 2020-04-02 日産化学株式会社 Additif pour milieu destiné à favoriser la production de facteur paracrine
WO2020158840A1 (fr) * 2019-01-30 2020-08-06 日産化学株式会社 Agent anticancéreux
WO2020158841A1 (fr) * 2019-01-30 2020-08-06 日産化学株式会社 Composé hydrazide et inhibiteur de kinase
WO2022085781A1 (fr) * 2020-10-23 2022-04-28 日産化学株式会社 Agent thérapeutique pour troubles hématopoïétiques

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