WO2022191296A1 - Medium composition, method for producing cell culture, and cell suspension - Google Patents
Medium composition, method for producing cell culture, and cell suspension Download PDFInfo
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- WO2022191296A1 WO2022191296A1 PCT/JP2022/010710 JP2022010710W WO2022191296A1 WO 2022191296 A1 WO2022191296 A1 WO 2022191296A1 JP 2022010710 W JP2022010710 W JP 2022010710W WO 2022191296 A1 WO2022191296 A1 WO 2022191296A1
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- medium
- medium composition
- cells
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Classifications
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- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present disclosure relates to medium compositions, methods for producing cell cultures, and cell suspensions.
- Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have proliferative potential and differentiation potential, and are therefore used in cell therapy and regenerative medicine. It is promising as a raw material for formulations administered by e.g. In order for cell therapy and regenerative medicine to be put into practical use, it is required that the necessary amount of pluripotent stem cells, which are raw materials, be stably supplied.
- the composition of the medium composition has a great impact on cell productivity.
- Components contained in the medium composition are selected according to the cell type or the purpose of culture.
- iPS cells and cardiomyocytes differentiated from iPS cells were co-cultured in a culture medium containing high concentrations of lactic acid. It has been reported that the cardiomyocytes derived from iPS cells can be purified because the cardiomyocytes selectively proliferate in this case.
- iSCIENCE, 2021, DOI: 10.1016/j.isci.2021.102090 reports that in undifferentiated maintenance culture of iPS cells, increasing the concentration of tryptophan in the culture medium improves the proliferation of iPS cells.
- the medium be a completely synthetic medium.
- pluripotent stem cells typified by ES cells and iPS cells have the problem of optimizing the medium composition for the purpose of improving the culture efficiency.
- the present disclosure provides a medium composition that can improve cell growth efficiency.
- the present disclosure provides a method for producing a cell culture capable of efficiently proliferating cells.
- the present disclosure provides cell suspensions that can efficiently grow cells.
- One embodiment contains at least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. , relating to the medium composition.
- Another embodiment relates to a method for producing a cell culture, comprising culturing cells using the medium composition.
- Other embodiments relate to cell suspensions containing the above media compositions and pluripotent stem cells.
- a medium composition capable of improving cell growth efficiency is obtained.
- ADVANTAGE OF THE INVENTION According to this indication, the manufacturing method of the cell culture which can proliferate a cell efficiently is obtained.
- a cell suspension is obtained that allows cells to grow efficiently.
- a numerical range indicated using “to” indicates a range including the numerical values before and after “to” as the minimum and maximum values, respectively.
- the upper limit value or lower limit value of the numerical range in one step can be arbitrarily combined with the upper limit value or lower limit of the numerical range in another step.
- the materials exemplified in this specification can be used singly or in combination of two or more unless otherwise specified.
- the content of each component in the composition refers to the total amount of the multiple substances present in the composition when there are multiple substances corresponding to each component in the composition, unless otherwise specified. means The term "process” is included in the term not only as an independent process, but also as long as the intended action of the process is achieved even if it is not clearly distinguishable from other processes.
- the medium composition contains at least one substance selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. contains.
- the at least one substance is effective for improving the growth efficiency of culture objects.
- the medium composition may contain, in addition to the at least one substance, known components contained in general medium compositions.
- the medium composition may be a liquid medium, a solid medium such as a powder medium, or a semi-solid medium such as a gel (soft agar) medium.
- a liquid medium can contain water as a base.
- a powder medium can be used as a liquid medium or a gel medium by combining with water or the like at the time of use.
- a powder medium can be obtained by a known method such as drying after preparation of a medium composition containing an aqueous medium.
- the aminoalkylsulfonic acid may be a compound represented by NR' 2 --R--SO 3 H (R represents an alkylene group and each R' independently represents a hydrogen atom or an alkyl group).
- Aminoalkylsulfonates are compounds represented by NR' 2 -R - SO 3 -X + (R represents an alkylene group, R' each independently represents a hydrogen atom or an alkyl group, X + , represents a cation.).
- the alkylene group can be a linear alkylene group, a branched alkylene group, or a cyclic alkylene group.
- the number of carbon atoms in the alkylene group may be, for example, 1-8, or 1-4.
- Alkylene groups are, for example, methylene group, ethylene group, n-propylene group, n-butylene group, n-pentylene group, n-hexylene group, n-heptylene group, n-octylene group, isopropylene group, isobutylene group, sec -butylene group, tert-butylene group, 2-ethylhexylene group, 3,7-dimethyloctylene group, cyclohexylene group, cycloheptylene group, and cyclooctylene group.
- Alkyl groups may be straight chain alkyl groups, branched alkyl groups, or cyclic alkyl groups. The number of carbon atoms in the alkyl group may be, for example, 1-8, 1-4, or 1 or 2.
- Alkyl groups include, for example, methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, isopropyl group, isobutyl group, sec- It contains at least one selected from the group consisting of a butyl group, a tert-butyl group, a 2-ethylhexyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.
- X + contains at least one selected from the group consisting of sodium ions, potassium ions, and am
- Aminoalkylsulfonic acids are, for example, aminomethylsulfonic acid, 2-aminoethylsulfonic acid, 2-(N-methylamino)ethylsulfonic acid, 2-(N-ethylamino)ethylsulfonic acid, 3-aminopropylsulfonic acid , 4-aminobutylsulfonic acid, 5-aminopentylsulfonic acid, 6-aminohexylsulfonic acid, 7-aminoheptylsulfonic acid, and 8-aminooctylsulfonic acid.
- the aminoalkylsulfonate includes, for example, at least one selected from the group consisting of sodium aminoalkylsulfonate, potassium aminoalkylsulfonate, and ammonium aminoalkylsulfonate.
- Aminoalkylsulfonates are, for example, aminomethylsulfonate, 2-aminoethylsulfonate, 2-(N-methylamino)ethylsulfonate, 2-(N-ethylamino)ethylsulfonate, 3 -aminopropylsulfonate, 4-aminobutylsulfonate, 5-aminopentylsulfonate, 6-aminohexylsulfonate, 7-aminoheptylsulfonate, and 8-aminooctylsulfonate and at least one selected from the group consisting of sodium ions, potassium ions, and ammonium ions.
- the content of the aminoalkylsulfonic acid in the medium composition is, for example, 50 to 200 ⁇ g relative to the total volume of the medium composition when used as a liquid medium or gel medium. /mL, 100-150 ⁇ g/mL, or 110-140 ⁇ g/mL.
- the medium composition is a powder medium
- the liquid medium or gel medium prepared from the powder medium may satisfy the above range of aminoalkylsulfonic acid content.
- the content of the aminoalkylsulfonate should satisfy the range of the content of aminoalkylsulfonic acid described above when the cations are replaced with hydrogen ions.
- the total content should satisfy the above content range.
- the iron carboxylate may be a salt containing a —COO — group as an anion, an iron ion and a cation other than iron ion as cations, and may be a salt containing R—(COO ⁇ )n as an anion and iron ion and iron as cations. It may be an organic ammonium iron carboxylate containing cations other than ions (R represents an organic group with a valence of 1 or more, n represents an integer of 1 or more. n is a —COO — group bonded to R is the number of ). Cations other than iron ions include, for example, at least one selected from the group consisting of sodium ions, potassium ions, and ammonium ions.
- the iron carboxylate preferably contains at least one selected from the group consisting of ammonium iron carboxylate and sodium iron carboxylate, and more preferably contains ammonium iron carboxylate.
- the ferric ammonium carboxylate may be a salt containing a —COO — group as anion and iron and ammonium ions as cations, wherein R—(COO ⁇ )n as anion and iron and ammonium ions as cations.
- R represents an organic group with a valence of 1 or more
- n represents an integer of 1 or more
- n is the number of —COO — groups bonded to R.
- R is an atomic group obtained by removing one or more hydrogen atoms from a substituted or unsubstituted saturated hydrocarbon
- the saturated hydrocarbon includes linear saturated hydrocarbon, branched saturated It may be a hydrocarbon or a saturated cyclic hydrocarbon.
- the saturated hydrocarbon may have, for example, 1 to 12 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms.
- Saturated hydrocarbons include, for example, at least one selected from the group consisting of methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane.
- Substituents that the saturated hydrocarbon may have include, for example, a hydroxy group, an amino group, a monoalkylamino group (the number of carbon atoms in the alkyl is, for example, 1 to 4), and a dialkylamino group (the number of carbon atoms in the alkyl is each independently, for example, 1 to 4.) and the like.
- n can be, for example, 2-6, or 2-5.
- Ferric ammonium carboxylates are, for example, ferric ammonium citrate, ferric ammonium pyruvate, ferric ammonium oxalate, ferric ammonium fumarate, ferric ammonium maleate, ferric ammonium malate, ferric ammonium succinate, diethylenetriamine ferric ammonium pentaacetate, ethylenediamine It contains at least one selected from the group consisting of ferric ammonium tetraacetate and ferric ammonium dicarboxymethylglutamate.
- the medium composition may contain sodium ferric carboxylate instead of or together with ferric ammonium carboxylate.
- the content of the iron carboxylate in the medium composition is the following iron ammonium carboxylate when the mass of anions other than iron is replaced by the mass of ammonium ions. It is sufficient if the content range is satisfied.
- the content of ammonium ferric carboxylate in the medium composition is, for example, 0.1 with respect to the total volume of the medium composition when used as a liquid medium or gel medium. It may be ⁇ 1 ⁇ g/mL, 0.2-0.5 ⁇ g/mL, or 0.2-0.4 ⁇ g/mL.
- the medium composition is a powder medium
- the liquid medium or gel medium prepared from the powder medium may satisfy the above range of the content of ferric ammonium carboxylate.
- the content of sodium iron carboxylate may satisfy the range of the content of ammonium iron carboxylate when sodium ions are replaced with ammonium ions.
- the content of dimethyloxalylglycine in the medium composition is, for example, 1 pg/mL to 1 ⁇ g with respect to the total volume of the medium composition when used as a liquid medium or gel medium. /mL, 10 pg/mL to 100 ng/mL, 100 pg/mL to 10 ng/mL, or 500 pg/mL to 1 ng/mL.
- the medium composition is a powder medium
- the liquid medium or gel medium prepared from the powder medium may satisfy the above range of dimethyloxalylglycine content.
- the content of 4-phenylbutyric acid in the medium composition is, for example, 0.001 with respect to the total volume of the medium composition when used as a liquid medium or gel medium. It may be ⁇ 1000 ⁇ g/mL, 0.01-100 ⁇ g/mL, 0.1-30 ⁇ g/mL, or 1-5 ⁇ g/mL.
- the medium composition is a powder medium
- the liquid medium or gel medium prepared from the powder medium may satisfy the above range of 4-phenylbutyric acid content.
- the content of 4-phenylbutyrate should satisfy the range of the content of 4-phenylbutyric acid described above when the cations are replaced with hydrogen ions.
- the medium composition contains 4-phenylbutyric acid and 4-phenylbutyrate, the total content should satisfy the above content range.
- the cation contained in 4-phenylbutyrate includes, for example, at least one selected from the group consisting of sodium ion, potassium ion, and ammonium ion.
- the medium composition contains two or more selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate.
- aspects of the medium composition include the following aspects. - A medium composition containing an aminoalkylsulfonic acid and an iron carboxylate - A medium composition containing an aminoalkylsulfonate and an iron carboxylate - A medium composition containing dimethyloxalylglycine and 4-phenylbutyric acid A medium composition containing dimethyloxalylglycine and 4-phenylbutyrate
- the medium composition may be a medium containing heterologous raw materials or a completely synthetic medium containing no heterologous raw materials. Also when the medium composition is a completely synthetic medium, it is selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. cells can be efficiently proliferated.
- inorganic salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium hydrogen carbonate, calcium nitrate, and iron nitrate.
- Sugars include, for example, glucose and sucrose.
- Amino acids include, for example, alanine, glycine, tryptophan, phenylalanine, tyrosine, arginine, histidine, lysine, aspartic acid, glutamic acid, asparagine, glutamine, leucine, isoleucine, valine, threonine, methionine, cysteine, proline, and the like.
- Proteins include, for example, plasma-derived proteins and the like.
- serum substitutes include bovine serum albumin (BSA), knockout serum replacement (KSR), human serum albumin (HSA or human albumin, serum; HAS), and the like. mentioned.
- vitamins include nicotinamide, ascorbic acid derivatives, riboflavin, pyridoxal, thiamine, folic acid and the like.
- Hormones include, for example, insulin, estradiol, progesterone, testosterone, thyroxine, and the like.
- antibiotics include penicillin, streptomycin and the like.
- Growth factors include, for example, epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2) and the like.
- biological tissue extracts include pituitary gland extracts and platelet extracts.
- the medium composition includes, for example, a known basal medium or other existing medium, aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate.
- basal media include DMEM (Dulbecco's modified Eagle's medium), MEM (Eagle's minimal essential medium), ⁇ MEM medium (Eagle's minimal essential medium ⁇ -modified), GMEM (Glasgow's minimal essential medium), IMDM (Iscove's modified Dulbecco's medium ), Ham's F12 (nutrient mixture F-12 ham), RPMI-1640 (RPMI-1640 medium), McCoy's 5A (McCoy's 5A medium), MSC growth medium 2 (Promocell), Prime XV XSFM (registered trademark) , Fuji Film Irvine Scientific), Essential 8 (Thermo Fisher Scientific), Essential 6 (Thermo Fisher Scientific), and mixtures containing two or more selected from these.
- DMEM Dynabecco's modified Eagle's medium
- MEM Eagle's minimal essential medium
- ⁇ MEM medium Eagle's minimal essential medium ⁇ -modified
- GMEM Gasgow'
- Essential 8 is a medium consisting of DMEM/F-12, L-ascorbic acid, selenium, transferrin, NaHCO 3 , insulin, FGF2 and TGF ⁇ 1, and water.
- Essential 6 is a medium consisting of DMEM/F-12, L-ascorbic acid, selenium, transferrin, NaHCO 3 and insulin, and water. Since Essential 8 and Essential 6 contain few components, they are easy to manage and can suppress variation between culture lots.
- the medium composition is not particularly limited and can be used for culturing various cells.
- Cells are preferably animal-derived cells, more preferably mammalian-derived cells. Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like.
- Cells include, for example, pluripotent stem cells, somatic stem cells, somatic cells, and the like.
- the cells may be pluripotent stem cells.
- Pluripotent stem cells include, for example, ES cells, iPS cells, EG cells (embryonic germ cells), and GS cells (germ-line stem cells).
- Somatic stem cells include mesenchymal stem cells, tissue stem cells, and the like.
- the medium composition is not particularly limited, and can be used as a medium for various culture methods or for preparing the medium.
- culture methods include two-dimensional culture in which cells are adhered to a fixed surface and cultured, and three-dimensional culture in which cells are cultured while suspended in a medium.
- cells may be in any form, may be suspended as single cells, cell clusters, or cell aggregates, and may be adhered to a culture carrier such as a microcarrier. You can float in the In the present disclosure, "single cell" means an independent cell.
- a method for producing a cell culture includes culturing cells using the medium composition according to the present disclosure.
- a liquid medium or a gel medium can be used as a medium composition for culture.
- the method for culturing the cells is not particularly limited, and the culture method described above can be used.
- a method for producing a cell culture includes culturing cells in suspension using a liquid medium as the medium composition.
- Cells to be cultured are also not particularly limited, and the cells described above can be cultured.
- a method for producing a cell culture includes culturing pluripotent stem cells using the medium composition. Thereby, a cell culture containing pluripotent stem cells and the medium composition according to the present disclosure can be obtained. That is, the cell culture according to the present disclosure includes the medium composition according to the present disclosure and cultured pluripotent stem cells.
- the culture temperature may be 36.0-38.0°C, or 36.5-37.5°C, regardless of the culture method.
- the culture atmosphere can be adjusted using a CO2 incubator or the like.
- the culture atmosphere may be a 1-10%, or 3-7% CO 2 atmosphere.
- a method for producing a cell culture may include any steps.
- Optional steps include, for example, thawing frozen cells, washing cells, seeding cells, adding medium or at least one component contained in medium to cell culture, cell culture exchanging at least part of the medium contained in the product, allowing the cell culture to stand, separating the cells from the medium composition using a filter or the like after a predetermined culture period, and collecting the collected Examples include preserving the cells in combination with a preserving composition.
- Preservation compositions include other known preservation media, cryopreservation compositions, and the like.
- a method for culturing cells includes culturing cells using the medium composition according to the present disclosure.
- the above description of the cell culture production method can also be applied to the cell culture method.
- a cell culture is obtained by a method for producing a cell culture or a method for culturing cells.
- a cell culture comprises a medium composition according to the present disclosure and cells. Proliferated cells can be recovered from the cultured cell culture.
- a cell suspension contains a medium composition according to the present disclosure and pluripotent stem cells.
- Pluripotent stem cells may be pluripotent stem cells cultured using the medium composition of the present disclosure.
- Cells obtained by culturing using the medium composition according to the present disclosure can have undifferentiation potential.
- the undifferentiated potential of cells can be confirmed by confirmation methods known in the art.
- Methods for confirming undifferentiated potency include, for example, methods for measuring the expression levels, abundance, and the like of known factors such as genes, proteins, and glycolipids, singly or in combination. Such factors are sometimes referred to as markers, and the genes and proteins used as markers are sometimes referred to as marker genes and marker proteins, respectively.
- Markers include Nanog, Sox2, Oct-4, Klf4, Lin28, TRA-1-60, TRA-1-81, TRA-2-49/6E, SSEA-1, SSEA-3, SSEA-4, c- and Myc. These markers can be used alone or in combination of two or more to evaluate the undifferentiated potential of cells. Specific methods for confirming the undifferentiated potential of cells using markers include a method of measuring the expression level of marker genes by quantitative PCR, etc., and the presence or absence of markers such as surface antigens by immunoassays such as flow cytometry. Alternatively, a method of confirming or measuring the abundance can be used.
- Cells with undifferentiated potential can be compared with known cells that do not exhibit undifferentiated potential depending on the cell type, origin, etc., and can be compared with markers such as Nanog, Sox2, Oct-4, Klf4, Lin28, TRA-1- 60, TRA-1-81, TRA-2-49/6E, SSEA-1, SSEA-3, SSEA-4, c-Myc, etc., for example Nanog, Sox2, Oct-4, There may be a trend towards increased abundance of one or more, such as SSEA-4.
- cells with undifferentiated potency are, for example, compared to known cells that do not show undifferentiated potency, due to the cell type, origin, etc., having a high Nanog amount, a high Sox2 amount, a high Oct-4 amount, a high Klf4 amount, high Lin28 amount, high TRA-1-60 amount, high TRA-1-81 amount, high TRA-2-49/6E amount, high SSEA-1 amount, high SSEA-3 amount, high SSEA-4 amount , high c-Myc amount, etc., for example, high Nanog amount, high Sox2 amount, high Oct-4 amount, high SSEA-4 amount. can do.
- Composition (2) The medium composition according to (1) above, which contains the aminoalkylsulfonic acid.
- (11) The medium composition according to any one of (1) to (10) above, which is used for culturing pluripotent stem cells. Alternatively, (11′) use of the medium composition according to any one of (1) to (10) above for culturing pluripotent stem cells.
- the culture may be suspension culture.
- (14) The method for producing a cell culture according to (12) or (13) above, wherein the cells comprise pluripotent stem cells.
- (15) A cell suspension containing the medium composition according to any one of (1) to (11) above and pluripotent stem cells.
- Example 1 Human iPS cells (strain 201B7) obtained from National University Corporation Kyoto University were used as cells.
- Example 2 To 6 mL of cell culture medium 1 prepared as described in Example 1, 1.5 ⁇ g of ferric ammonium citrate (Fuji Film Wako Pure Chemical Industries, Ltd.) was added to prepare cell culture medium 2. The amount of bFGF added was 100 ng/mL, the amount of 2-aminoethylsulfonic acid added was 125 ⁇ g/mL, and the amount of ferric ammonium citrate added was 0.25 ⁇ g/mL. Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells.
- ferric ammonium citrate Feji Film Wako Pure Chemical Industries, Ltd.
- Example 3 To 6 mL of Essential 6 (Thermo Fisher Scientific), 600 ng of bFGF (R&D Systems) and 1.5 ⁇ g of ferric ammonium citrate (Fuji Film Wako Pure Chemical Industries, Ltd.) were added to prepare a cell culture solution 3. The amount of ferric ammonium citrate added was 0.25 ⁇ g/mL. Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells. PDL was 3.38 on day 4 of culture.
- Comparative Example 2 Suspension culture was performed in the same manner as in Comparative Example 1, and PDL was calculated from the results of counting the number of viable cells. PDL was 2.78 on day 4 of culture.
- Example 4 (Preparation of cell culture medium (liquid medium)) Cell culture medium 4 was prepared by adding 5 nM of dimethyloxalylglycine (R&D systems) to 1.5 mL of DMEM/F12-based reference medium. The amount of dimethyloxalylglycine added was 0.88 ng/mL.
- Example 5 Cell culture medium 5 was prepared by adding 10 ⁇ M 4-phenylbutyric acid (Sigma) to 1.5 mL of DMEM/F12-based reference medium. The amount of 4-phenylbutyric acid added was 1.6 ⁇ g/mL. Subsequently, suspension culture was performed in the same manner as in Example 4, and PDL was calculated from the results of counting the number of viable cells. PDL was 2.29 on day 7 of culture.
- Example 3 Suspension culture was performed in the same manner as in Example 4, except that 1.5 mL of a DMEM/F12-based reference medium was used as the cell culture medium, and PDL was calculated from the results of viable cell counts. PDL was 1.69 on day 7 of culture.
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Abstract
The present disclosure pertains to a medium composition that contains at least one selected from the group consisting of an aminoalkylsulfonic acid, an aminoalkylsulfonic acid salt, an iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and a 4-phenylbutyric acid salt.
Description
本開示は、培地組成物、細胞培養物の製造方法、及び細胞懸濁物に関する。
The present disclosure relates to medium compositions, methods for producing cell cultures, and cell suspensions.
胚性幹細胞（Ｅｍｂｒｙｏｎｉｃ ｓｔｅｍ ｃｅｌｌ；ＥＳ細胞）及び人工多能性幹細胞（Ｉｎｄｕｃｅｄ ｐｌｕｒｉｐｏｔｅｎｔ ｓｔｅｍ ｃｅｌｌ；ｉＰＳ細胞）等の多能性幹細胞は、増殖能と分化能とを有することから、細胞治療及び再生医療等で投与される製剤の原料として、有望である。細胞治療及び再生医療が実用化されるには、原料である多能性幹細胞が、安定的に必要量供給されることが求められる。
Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have proliferative potential and differentiation potential, and are therefore used in cell therapy and regenerative medicine. It is promising as a raw material for formulations administered by e.g. In order for cell therapy and regenerative medicine to be put into practical use, it is required that the necessary amount of pluripotent stem cells, which are raw materials, be stably supplied.
産業的に細胞の培養を制御する上で、培地組成物の組成は、細胞の生産性に大きな影響を及ぼす。細胞種又は培養の目的に応じて、培地組成物に含まれる成分が選択される。
In industrially controlling cell culture, the composition of the medium composition has a great impact on cell productivity. Components contained in the medium composition are selected according to the cell type or the purpose of culture.
例えば、Cell Stem Cell, 2013, Vol. 12, (1), pp. 127-137では、高濃度の乳酸を含む培養液中で、ｉＰＳ細胞とｉＰＳ細胞から分化誘導した心筋細胞とを共培養した場合、心筋細胞が選択的に増殖することから、ｉＰＳ細胞由来の心筋細胞の純化が可能であることが報告されている。
For example, in Cell Stem Cell, 2013, Vol. 12, (1), pp. 127-137, iPS cells and cardiomyocytes differentiated from iPS cells were co-cultured in a culture medium containing high concentrations of lactic acid. It has been reported that the cardiomyocytes derived from iPS cells can be purified because the cardiomyocytes selectively proliferate in this case.
iSCIENCE, 2021, DOI: 10.1016/j.isci.2021.102090では、ｉＰＳ細胞の未分化維持培養において、培養液中のトリプトファン濃度を増やすと、ｉＰＳ細胞の増殖性が向上することが報告されている。
iSCIENCE, 2021, DOI: 10.1016/j.isci.2021.102090 reports that in undifferentiated maintenance culture of iPS cells, increasing the concentration of tryptophan in the culture medium improves the proliferation of iPS cells.
国際公開第２０１４／１１９２１９号では、多能性幹細胞の増殖促進に、エタノールアミンの添加が有効であることが開示されている。
International Publication No. 2014/119219 discloses that the addition of ethanolamine is effective in promoting the proliferation of pluripotent stem cells.
細胞治療及び再生医療の原料に用いられる細胞の培養には、安全性の観点から、培養工程で、異種原料を使用しないことが推奨されており、培地も完全合成培地であることが、望ましい。
From the perspective of safety, it is recommended not to use heterologous raw materials in the culture process for culturing cells used as raw materials for cell therapy and regenerative medicine, and it is desirable that the medium be a completely synthetic medium.
しかしながら、異種原料を使用した培地に比べ、完全合成培地での培養は、細胞の増殖効率の面で改善の余地がある。特に、ＥＳ細胞及びｉＰＳ細胞に代表される多能性幹細胞では、培養効率の向上を目的とした、培地の組成の至適化が望まれているという課題がある。
However, compared to media using heterologous raw materials, culturing in completely synthetic media has room for improvement in terms of cell growth efficiency. In particular, pluripotent stem cells typified by ES cells and iPS cells have the problem of optimizing the medium composition for the purpose of improving the culture efficiency.
本開示は、細胞の増殖効率を向上させることができる培地組成物を提供する。本開示は、効率よく細胞を増殖させることができる細胞培養物の製造方法を提供する。本開示は、効率よく細胞を増殖させることができる細胞懸濁物を提供する。
The present disclosure provides a medium composition that can improve cell growth efficiency. The present disclosure provides a method for producing a cell culture capable of efficiently proliferating cells. The present disclosure provides cell suspensions that can efficiently grow cells.
実施形態の例を以下に挙げる。本発明は以下の実施形態に限定されない。
一実施形態は、アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種を含有する、培地組成物に関する。
他の実施形態は、上記培地組成物を用いて、細胞を培養することを含む、細胞培養物の製造方法に関する。
他の実施形態は、上記培地組成物と、多能性幹細胞とを含有する、細胞懸濁物に関する。 Examples of embodiments are provided below. The present invention is not limited to the following embodiments.
One embodiment contains at least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. , relating to the medium composition.
Another embodiment relates to a method for producing a cell culture, comprising culturing cells using the medium composition.
Other embodiments relate to cell suspensions containing the above media compositions and pluripotent stem cells.
一実施形態は、アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種を含有する、培地組成物に関する。
他の実施形態は、上記培地組成物を用いて、細胞を培養することを含む、細胞培養物の製造方法に関する。
他の実施形態は、上記培地組成物と、多能性幹細胞とを含有する、細胞懸濁物に関する。 Examples of embodiments are provided below. The present invention is not limited to the following embodiments.
One embodiment contains at least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. , relating to the medium composition.
Another embodiment relates to a method for producing a cell culture, comprising culturing cells using the medium composition.
Other embodiments relate to cell suspensions containing the above media compositions and pluripotent stem cells.
本開示によれば、細胞の増殖効率を向上させることができる培地組成物が得られる。本開示によれば、効率よく細胞を増殖させることができる細胞培養物の製造方法が得られる。本開示によれば、効率よく細胞を増殖させることができる細胞懸濁物が得られる。
According to the present disclosure, a medium composition capable of improving cell growth efficiency is obtained. ADVANTAGE OF THE INVENTION According to this indication, the manufacturing method of the cell culture which can proliferate a cell efficiently is obtained. According to the present disclosure, a cell suspension is obtained that allows cells to grow efficiently.
本発明の実施形態について説明する。本発明は以下の実施形態に限定されない。本明細書において、「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。本明細書に段階的に記載されている数値範囲において、ある段階の数値範囲の上限値又は下限値は、他の段階の数値範囲の上限値又は下限値と任意に組み合わせることができる。本明細書に例示する材料は、特に断らない限り、１種を単独で又は２種以上を組み合わせて用いることができる。本明細書において、組成物中の各成分の含有量は、組成物中に各成分に該当する物質が複数存在する場合、特に断らない限り、組成物中に存在する当該複数の物質の合計量を意味する。「工程」との語は、独立した工程だけではなく、他の工程と明確に区別できない場合であってもその工程の所期の作用が達成されれば、本用語に含まれる。
An embodiment of the present invention will be described. The present invention is not limited to the following embodiments. In this specification, a numerical range indicated using "to" indicates a range including the numerical values before and after "to" as the minimum and maximum values, respectively. In the numerical ranges described stepwise in this specification, the upper limit value or lower limit value of the numerical range in one step can be arbitrarily combined with the upper limit value or lower limit of the numerical range in another step. The materials exemplified in this specification can be used singly or in combination of two or more unless otherwise specified. As used herein, the content of each component in the composition refers to the total amount of the multiple substances present in the composition when there are multiple substances corresponding to each component in the composition, unless otherwise specified. means The term "process" is included in the term not only as an independent process, but also as long as the intended action of the process is achieved even if it is not clearly distinguishable from other processes.
＜培地組成物＞
培地組成物は、アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種の物質を含有する。前記少なくとも１種の物質は、培養対象物の増殖効率の向上に有効である。培地組成物は、前記少なくとも１種の物質以外に、一般的な培地組成物に含まれる公知の成分を含有してよい。 <Medium composition>
The medium composition contains at least one substance selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. contains. The at least one substance is effective for improving the growth efficiency of culture objects. The medium composition may contain, in addition to the at least one substance, known components contained in general medium compositions.
培地組成物は、アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種の物質を含有する。前記少なくとも１種の物質は、培養対象物の増殖効率の向上に有効である。培地組成物は、前記少なくとも１種の物質以外に、一般的な培地組成物に含まれる公知の成分を含有してよい。 <Medium composition>
The medium composition contains at least one substance selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. contains. The at least one substance is effective for improving the growth efficiency of culture objects. The medium composition may contain, in addition to the at least one substance, known components contained in general medium compositions.
培地組成物は、液体培地、粉末培地等の固体培地、又は、ゲル（軟寒天）培地等の半固体培地であってよい。液体培地は、水を基剤として含有することができる。粉末培地は、使用時に水等と組み合わせて液体培地又はゲル培地として使用することができる。粉末培地は、水性媒体を含有する培地組成物を調製後に乾燥処理するなどの公知の方法によって得られる。
The medium composition may be a liquid medium, a solid medium such as a powder medium, or a semi-solid medium such as a gel (soft agar) medium. A liquid medium can contain water as a base. A powder medium can be used as a liquid medium or a gel medium by combining with water or the like at the time of use. A powder medium can be obtained by a known method such as drying after preparation of a medium composition containing an aqueous medium.
アミノアルキルスルホン酸は、ＮＲ’２－Ｒ－ＳＯ３Ｈで表される化合物（Ｒは、アルキレン基を表し、Ｒ’は、それぞれ独立に水素原子又はアルキル基を表す。）であってよい。アミノアルキルスルホン酸塩は、ＮＲ’２－Ｒ－ＳＯ３
－Ｘ＋で表される化合物（Ｒは、アルキレン基を表し、Ｒ’は、それぞれ独立に水素原子又はアルキル基を表し、Ｘ＋は、カチオンを表す。）であってよい。
The aminoalkylsulfonic acid may be a compound represented by NR' 2 --R--SO 3 H (R represents an alkylene group and each R' independently represents a hydrogen atom or an alkyl group). Aminoalkylsulfonates are compounds represented by NR' 2 -R - SO 3 -X + (R represents an alkylene group, R' each independently represents a hydrogen atom or an alkyl group, X + , represents a cation.).
アルキレン基は、直鎖アルキレン基、分岐アルキレン基、又は環状アルキレン基であってよい。アルキレン基の炭素数は、例えば、１～８であってよく、１～４であってよい。
アルキレン基は、例えば、メチレン基、エチレン基、ｎ－プロピレン基、ｎ－ブチレン基、ｎ－ペンチレン基、ｎ－ヘキシレン基、ｎ－ヘプチレン基、ｎ－オクチレン基、イソプロピレン基、イソブチレン基、ｓｅｃ－ブチレン基、ｔｅｒｔ－ブチレン基、２－エチルヘキシレン基、３，７－ジメチルオクチレン基、シクロヘキシレン基、シクロヘプチレン基、及びシクロオクチレン基からなる群から選択される少なくとも１種を含む。アルキル基は、直鎖アルキル基、分岐アルキル基、又は環状アルキル基であってよい。アルキル基の炭素数は、例えば、１～８であってよく、１～４であってよく、１又は２であってよい。アルキル基は、例えば、メチル基、エチル基、ｎ－プロピル基、ｎ－ブチル基、ｎ－ペンチル基、ｎ－ヘキシル基、ｎ－ヘプチル基、ｎ－オクチル基、イソプロピル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、２－エチルヘキシル基、シクロヘキシル基、シクロヘプチル基、及びシクロオクチル基からなる群から選択される少なくとも１種を含む。Ｘ＋は、ナトリウムイオン、カリウムイオン、及びアンモニウムイオンからなる群から選択される少なくとも１種を含む。 The alkylene group can be a linear alkylene group, a branched alkylene group, or a cyclic alkylene group. The number of carbon atoms in the alkylene group may be, for example, 1-8, or 1-4.
Alkylene groups are, for example, methylene group, ethylene group, n-propylene group, n-butylene group, n-pentylene group, n-hexylene group, n-heptylene group, n-octylene group, isopropylene group, isobutylene group, sec -butylene group, tert-butylene group, 2-ethylhexylene group, 3,7-dimethyloctylene group, cyclohexylene group, cycloheptylene group, and cyclooctylene group. Alkyl groups may be straight chain alkyl groups, branched alkyl groups, or cyclic alkyl groups. The number of carbon atoms in the alkyl group may be, for example, 1-8, 1-4, or 1 or 2. Alkyl groups include, for example, methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, isopropyl group, isobutyl group, sec- It contains at least one selected from the group consisting of a butyl group, a tert-butyl group, a 2-ethylhexyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group. X + contains at least one selected from the group consisting of sodium ions, potassium ions, and ammonium ions.
アルキレン基は、例えば、メチレン基、エチレン基、ｎ－プロピレン基、ｎ－ブチレン基、ｎ－ペンチレン基、ｎ－ヘキシレン基、ｎ－ヘプチレン基、ｎ－オクチレン基、イソプロピレン基、イソブチレン基、ｓｅｃ－ブチレン基、ｔｅｒｔ－ブチレン基、２－エチルヘキシレン基、３，７－ジメチルオクチレン基、シクロヘキシレン基、シクロヘプチレン基、及びシクロオクチレン基からなる群から選択される少なくとも１種を含む。アルキル基は、直鎖アルキル基、分岐アルキル基、又は環状アルキル基であってよい。アルキル基の炭素数は、例えば、１～８であってよく、１～４であってよく、１又は２であってよい。アルキル基は、例えば、メチル基、エチル基、ｎ－プロピル基、ｎ－ブチル基、ｎ－ペンチル基、ｎ－ヘキシル基、ｎ－ヘプチル基、ｎ－オクチル基、イソプロピル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、２－エチルヘキシル基、シクロヘキシル基、シクロヘプチル基、及びシクロオクチル基からなる群から選択される少なくとも１種を含む。Ｘ＋は、ナトリウムイオン、カリウムイオン、及びアンモニウムイオンからなる群から選択される少なくとも１種を含む。 The alkylene group can be a linear alkylene group, a branched alkylene group, or a cyclic alkylene group. The number of carbon atoms in the alkylene group may be, for example, 1-8, or 1-4.
Alkylene groups are, for example, methylene group, ethylene group, n-propylene group, n-butylene group, n-pentylene group, n-hexylene group, n-heptylene group, n-octylene group, isopropylene group, isobutylene group, sec -butylene group, tert-butylene group, 2-ethylhexylene group, 3,7-dimethyloctylene group, cyclohexylene group, cycloheptylene group, and cyclooctylene group. Alkyl groups may be straight chain alkyl groups, branched alkyl groups, or cyclic alkyl groups. The number of carbon atoms in the alkyl group may be, for example, 1-8, 1-4, or 1 or 2. Alkyl groups include, for example, methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, isopropyl group, isobutyl group, sec- It contains at least one selected from the group consisting of a butyl group, a tert-butyl group, a 2-ethylhexyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group. X + contains at least one selected from the group consisting of sodium ions, potassium ions, and ammonium ions.
アミノアルキルスルホン酸は、例えば、アミノメチルスルホン酸、２－アミノエチルスルホン酸、２－（Ｎ－メチルアミノ）エチルスルホン酸、２－（Ｎ－エチルアミノ）エチルスルホン酸、３－アミノプロピルスルホン酸、４－アミノブチルスルホン酸、５－アミノペンチルスルホン酸、６－アミノヘキシルスルホン酸、７－アミノヘプチルスルホン酸、及び８－アミノオクチルスルホン酸からなる群から選択される少なくとも１種を含む。
Aminoalkylsulfonic acids are, for example, aminomethylsulfonic acid, 2-aminoethylsulfonic acid, 2-(N-methylamino)ethylsulfonic acid, 2-(N-ethylamino)ethylsulfonic acid, 3-aminopropylsulfonic acid , 4-aminobutylsulfonic acid, 5-aminopentylsulfonic acid, 6-aminohexylsulfonic acid, 7-aminoheptylsulfonic acid, and 8-aminooctylsulfonic acid.
アミノアルキルスルホン酸塩は、例えば、アミノアルキルスルホン酸ナトリウム、アミノアルキルスルホン酸カリウム、及びアミノアルキルスルホン酸アンモニウムからなる群から選択される少なくとも１種を含む。アミノアルキルスルホン酸塩は、例えば、アミノメチルスルホン酸イオン、２－アミノエチルスルホン酸イオン、２－（Ｎ－メチルアミノ）エチルスルホン酸イオン、２－（Ｎ－エチルアミノ）エチルスルホン酸イオン、３－アミノプロピルスルホン酸イオン、４－アミノブチルスルホン酸イオン、５－アミノペンチルスルホン酸イオン、６－アミノヘキシルスルホン酸イオン、７－アミノヘプチルスルホン酸イオン、及び８－アミノオクチルスルホン酸イオンからなる群から選択される少なくとも１種と、ナトリウムイオン、カリウムイオン、及びアンモニウムイオンからなる群から選択される少なくとも１種とを含む。
The aminoalkylsulfonate includes, for example, at least one selected from the group consisting of sodium aminoalkylsulfonate, potassium aminoalkylsulfonate, and ammonium aminoalkylsulfonate. Aminoalkylsulfonates are, for example, aminomethylsulfonate, 2-aminoethylsulfonate, 2-(N-methylamino)ethylsulfonate, 2-(N-ethylamino)ethylsulfonate, 3 -aminopropylsulfonate, 4-aminobutylsulfonate, 5-aminopentylsulfonate, 6-aminohexylsulfonate, 7-aminoheptylsulfonate, and 8-aminooctylsulfonate and at least one selected from the group consisting of sodium ions, potassium ions, and ammonium ions.
培地組成物がアミノアルキルスルホン酸を含有する場合、培地組成物におけるアミノアルキルスルホン酸の含有量は、液体培地又はゲル培地としたときに、培地組成物の全容量に対し、例えば、５０～２００μｇ／ｍＬ、１００～１５０μｇ／ｍＬ、又は１１０～１４０μｇ／ｍＬであってよい。培地組成物が粉末培地である場合、粉末培地から調製した液体培地又はゲル培地が、前記のアミノアルキルスルホン酸の含有量の範囲を満たせばよい。
When the medium composition contains aminoalkylsulfonic acid, the content of the aminoalkylsulfonic acid in the medium composition is, for example, 50 to 200 μg relative to the total volume of the medium composition when used as a liquid medium or gel medium. /mL, 100-150 μg/mL, or 110-140 μg/mL. When the medium composition is a powder medium, the liquid medium or gel medium prepared from the powder medium may satisfy the above range of aminoalkylsulfonic acid content.
培地組成物がアミノアルキルスルホン酸塩を含有する場合、アミノアルキルスルホン酸塩の含有量は、カチオンを水素イオンに置き換えた場合に、前記のアミノアルキルスルホン酸の含有量の範囲を満たせばよい。培地組成物がアミノアルキルスルホン酸とアミノアルキルスルホン酸塩とを含有する場合、合計の含有量が、前記の含有量の範囲を満たせばよい。
When the medium composition contains an aminoalkylsulfonate, the content of the aminoalkylsulfonate should satisfy the range of the content of aminoalkylsulfonic acid described above when the cations are replaced with hydrogen ions. When the medium composition contains an aminoalkylsulfonic acid and an aminoalkylsulfonate, the total content should satisfy the above content range.
カルボン酸鉄塩は、アニオンとして－ＣＯＯ－基と、カチオンとして鉄イオンと鉄イオン以外のカチオンとを含む塩であってよく、アニオンとしてＲ－（ＣＯＯ－）ｎと、カチオンとして鉄イオンと鉄イオン以外のカチオンとを含む有機カルボン酸鉄アンモニウムであってよい（Ｒは、１価以上の有機基を表し、ｎは、１以上の整数を表す。ｎは、Ｒに結合する－ＣＯＯ－基の個数である。）。鉄イオン以外のカチオンは、例えば、ナトリウムイオン、カリウムイオン、及びアンモニウムイオンからなる群から選択される少なくとも１種を含む。カルボン酸鉄塩は、カルボン酸鉄アンモニウム及びカルボン酸鉄ナトリウムからなる群から選択される少なくとも１種を含むことが好ましく、カルボン酸鉄アンモニウムを含むことがより好ましい。
The iron carboxylate may be a salt containing a —COO — group as an anion, an iron ion and a cation other than iron ion as cations, and may be a salt containing R—(COO − )n as an anion and iron ion and iron as cations. It may be an organic ammonium iron carboxylate containing cations other than ions (R represents an organic group with a valence of 1 or more, n represents an integer of 1 or more. n is a —COO — group bonded to R is the number of ). Cations other than iron ions include, for example, at least one selected from the group consisting of sodium ions, potassium ions, and ammonium ions. The iron carboxylate preferably contains at least one selected from the group consisting of ammonium iron carboxylate and sodium iron carboxylate, and more preferably contains ammonium iron carboxylate.
カルボン酸鉄アンモニウムは、アニオンとして－ＣＯＯ－基と、カチオンとして鉄イオン及びアンモニウムイオンとを含む塩であってよく、アニオンとしてＲ－（ＣＯＯ－）ｎと、カチオンとして鉄イオン及びアンモニウムイオンとを含む有機カルボン酸鉄アンモニウムであってよい（Ｒは、１価以上の有機基を表し、ｎは、１以上の整数を表す。ｎは、Ｒに結合する－ＣＯＯ－基の個数である。）。
The ferric ammonium carboxylate may be a salt containing a —COO — group as anion and iron and ammonium ions as cations, wherein R—(COO − )n as anion and iron and ammonium ions as cations. (R represents an organic group with a valence of 1 or more, n represents an integer of 1 or more, n is the number of —COO — groups bonded to R.) .
Ｒ－（ＣＯＯ－）ｎにおいて、Ｒは、置換又は非置換の飽和炭化水素から１個以上の水素原子を除いた原子団であり、飽和炭化水素は、直鎖状飽和炭化水素、分岐状飽和炭化水素、又は環状飽和炭化水素であってよい。飽和炭化水素の炭素数は、例えば、１～１２であってよく、１～８であってよく、１～６であってよい。飽和炭化水素は、例えば、メタン、エタン、プロパン、ブタン、ペンタン、ヘキサン、ヘプタン、オクタン、ノナン、デカン、ウンデカン、及びドデカンからなる群から選択される少なくとも１種を含む。飽和炭化水素が有してよい置換基として、例えば、ヒドロキシ基、アミノ基、モノアルキルアミノ基（アルキルの炭素数は、例えば１～４である。）、ジアルキルアミノ基（アルキルの炭素数は、それぞれ独立に、例えば、１～４である。）等が挙げられる。ｎは、例えば、２～６、又は２～５であってよい。
In R—(COO − )n, R is an atomic group obtained by removing one or more hydrogen atoms from a substituted or unsubstituted saturated hydrocarbon, and the saturated hydrocarbon includes linear saturated hydrocarbon, branched saturated It may be a hydrocarbon or a saturated cyclic hydrocarbon. The saturated hydrocarbon may have, for example, 1 to 12 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms. Saturated hydrocarbons include, for example, at least one selected from the group consisting of methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane. Substituents that the saturated hydrocarbon may have include, for example, a hydroxy group, an amino group, a monoalkylamino group (the number of carbon atoms in the alkyl is, for example, 1 to 4), and a dialkylamino group (the number of carbon atoms in the alkyl is each independently, for example, 1 to 4.) and the like. n can be, for example, 2-6, or 2-5.
カルボン酸鉄アンモニウムは、例えば、クエン酸鉄アンモニウム、ピルビン酸鉄アンモニウム、シュウ酸鉄アンモニウム、フマル酸鉄アンモニウム、マレイン酸鉄アンモニウム、リンゴ酸鉄アンモニウム、コハク酸鉄アンモニウム、ジエチレントリアミン五酢酸鉄アンモニウム、エチレンジアミン四酢酸鉄アンモニウム、及びジカルボキシメチルグルタミン酸鉄アンモニウムからなる群から選択される少なくとも１種を含む。培地組成物は、カルボン酸鉄アンモニウムに代えて、又は、カルボン酸鉄アンモニウムと共に、カルボン酸鉄ナトリウムを含有してもよい。
Ferric ammonium carboxylates are, for example, ferric ammonium citrate, ferric ammonium pyruvate, ferric ammonium oxalate, ferric ammonium fumarate, ferric ammonium maleate, ferric ammonium malate, ferric ammonium succinate, diethylenetriamine ferric ammonium pentaacetate, ethylenediamine It contains at least one selected from the group consisting of ferric ammonium tetraacetate and ferric ammonium dicarboxymethylglutamate. The medium composition may contain sodium ferric carboxylate instead of or together with ferric ammonium carboxylate.
培地組成物がカルボン酸鉄塩を含有する場合、培地組成物におけるカルボン酸鉄塩の含有量は、鉄以外のアニオンの質量をアンモニウムイオンの質量に置き換えた場合に、下記のカルボン酸鉄アンモニウムの含有量の範囲を満たせばよい。
When the medium composition contains an iron carboxylate, the content of the iron carboxylate in the medium composition is the following iron ammonium carboxylate when the mass of anions other than iron is replaced by the mass of ammonium ions. It is sufficient if the content range is satisfied.
培地組成物がカルボン酸鉄アンモニウムを含有する場合、培地組成物におけるカルボン酸鉄アンモニウムの含有量は、液体培地又はゲル培地としたときに、培地組成物の全容量に対し、例えば、０．１～１μｇ／ｍＬ、０．２～０．５μｇ／ｍＬ、又は０．２～０．４μｇ／ｍＬであってよい。培地組成物が粉末培地である場合、粉末培地から調製した液体培地又はゲル培地が、前記のカルボン酸鉄アンモニウムの含有量の範囲を満たせばよい。
When the medium composition contains ammonium ferric carboxylate, the content of ammonium ferric carboxylate in the medium composition is, for example, 0.1 with respect to the total volume of the medium composition when used as a liquid medium or gel medium. It may be ˜1 μg/mL, 0.2-0.5 μg/mL, or 0.2-0.4 μg/mL. When the medium composition is a powder medium, the liquid medium or gel medium prepared from the powder medium may satisfy the above range of the content of ferric ammonium carboxylate.
培地組成物がカルボン酸鉄ナトリウムを含有する場合、カルボン酸鉄ナトリウムの含有量は、ナトリウムイオンをアンモニウムイオンに置き換えた場合に、前記のカルボン酸鉄アンモニウムの含有量の範囲を満たせばよい。
When the medium composition contains sodium iron carboxylate, the content of sodium iron carboxylate may satisfy the range of the content of ammonium iron carboxylate when sodium ions are replaced with ammonium ions.
培地組成物がジメチルオキサリルグリシンを含有する場合、培地組成物におけるジメチルオキサリルグリシンの含有量は、液体培地又はゲル培地としたときに、培地組成物の全容量に対し、例えば、１ｐｇ／ｍＬ～１μｇ／ｍＬ、１０ｐｇ／ｍＬ～１００ｎｇ／ｍＬ、１００ｐｇ／ｍＬ～１０ｎｇ／ｍＬ、又は、５００ｐｇ／ｍＬ～１ｎｇ／ｍＬであってよい。培地組成物が粉末培地である場合、粉末培地から調製した液体培地又はゲル培地が、前記のジメチルオキサリルグリシンの含有量の範囲を満たせばよい。
When the medium composition contains dimethyloxalylglycine, the content of dimethyloxalylglycine in the medium composition is, for example, 1 pg/mL to 1 μg with respect to the total volume of the medium composition when used as a liquid medium or gel medium. /mL, 10 pg/mL to 100 ng/mL, 100 pg/mL to 10 ng/mL, or 500 pg/mL to 1 ng/mL. When the medium composition is a powder medium, the liquid medium or gel medium prepared from the powder medium may satisfy the above range of dimethyloxalylglycine content.
培地組成物が４－フェニル酪酸を含有する場合、培地組成物における４－フェニル酪酸の含有量は、液体培地又はゲル培地としたときに、培地組成物の全容量に対し、例えば、０．００１～１０００μｇ／ｍＬ、０．０１～１００μｇ／ｍＬ、０．１～３０μｇ／ｍＬ、又は、１～５μｇ／ｍＬであってよい。培地組成物が粉末培地である場合、粉末培地から調製した液体培地又はゲル培地が、前記の４－フェニル酪酸の含有量の範囲を満たせばよい。
When the medium composition contains 4-phenylbutyric acid, the content of 4-phenylbutyric acid in the medium composition is, for example, 0.001 with respect to the total volume of the medium composition when used as a liquid medium or gel medium. It may be ˜1000 μg/mL, 0.01-100 μg/mL, 0.1-30 μg/mL, or 1-5 μg/mL. When the medium composition is a powder medium, the liquid medium or gel medium prepared from the powder medium may satisfy the above range of 4-phenylbutyric acid content.
培地組成物が４－フェニル酪酸塩を含有する場合、４－フェニル酪酸塩の含有量は、カチオンを水素イオンに置き換えた場合に、前記の４－フェニル酪酸の含有量の範囲を満たせばよい。培地組成物が４－フェニル酪酸と４－フェニル酪酸塩とを含有する場合、合計の含有量が、前記含有量の範囲を満たせばよい。４－フェニル酪酸塩に含まれるカチオンは、例えば、ナトリウムイオン、カリウムイオン、及びアンモニウムイオンからなる群から選択される少なくとも１種を含む。
When the medium composition contains 4-phenylbutyrate, the content of 4-phenylbutyrate should satisfy the range of the content of 4-phenylbutyric acid described above when the cations are replaced with hydrogen ions. When the medium composition contains 4-phenylbutyric acid and 4-phenylbutyrate, the total content should satisfy the above content range. The cation contained in 4-phenylbutyrate includes, for example, at least one selected from the group consisting of sodium ion, potassium ion, and ammonium ion.
培地組成物は、アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される２種以上を含有してよい。例えば、培地組成物の態様として、以下の態様が挙げられる。
・アミノアルキルスルホン酸とカルボン酸鉄塩とを含有する培地組成物
・アミノアルキルスルホン酸塩とカルボン酸鉄塩とを含有する培地組成物
・ジメチルオキサリルグリシンと４－フェニル酪酸とを含有する培地組成物
・ジメチルオキサリルグリシンと４－フェニル酪酸塩とを含有する培地組成物 The medium composition contains two or more selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. you can For example, aspects of the medium composition include the following aspects.
- A medium composition containing an aminoalkylsulfonic acid and an iron carboxylate - A medium composition containing an aminoalkylsulfonate and an iron carboxylate - A medium composition containing dimethyloxalylglycine and 4-phenylbutyric acid A medium composition containing dimethyloxalylglycine and 4-phenylbutyrate
・アミノアルキルスルホン酸とカルボン酸鉄塩とを含有する培地組成物
・アミノアルキルスルホン酸塩とカルボン酸鉄塩とを含有する培地組成物
・ジメチルオキサリルグリシンと４－フェニル酪酸とを含有する培地組成物
・ジメチルオキサリルグリシンと４－フェニル酪酸塩とを含有する培地組成物 The medium composition contains two or more selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. you can For example, aspects of the medium composition include the following aspects.
- A medium composition containing an aminoalkylsulfonic acid and an iron carboxylate - A medium composition containing an aminoalkylsulfonate and an iron carboxylate - A medium composition containing dimethyloxalylglycine and 4-phenylbutyric acid A medium composition containing dimethyloxalylglycine and 4-phenylbutyrate
培地組成物が含むことができる任意の添加成分として、無機塩、糖、アミノ酸、タンパク質、血清、血清代替物、ビタミン、ホルモン、抗生物質、成長因子、生体組織抽出物等が挙げられる。培地組成物は、異種原料を含む培地であってもよく、異種原料を含まない完全合成培地であってもよい。培地組成物が完全合成培地である場合も、アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種を含むために、細胞を効率よく増殖させることができる。
Inorganic salts, sugars, amino acids, proteins, serum, serum substitutes, vitamins, hormones, antibiotics, growth factors, biological tissue extracts, etc., can be mentioned as optional additive components that the medium composition can contain. The medium composition may be a medium containing heterologous raw materials or a completely synthetic medium containing no heterologous raw materials. Also when the medium composition is a completely synthetic medium, it is selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. cells can be efficiently proliferated.
無機塩としては、例えば、塩化リチウム、塩化ナトリウム、塩化カリウム、塩化カルシウム、リン酸ナトリウム、リン酸一水素ナトリウム、リン酸二水素ナトリウム、炭酸水素ナトリウム、硝酸カルシウム、硝酸鉄等が挙げられる。糖としては、例えば、グルコース、スクロース等が挙げられる。アミノ酸としては、例えば、アラニン、グリシン、トリプトファン、フェニルアラニン、チロシン、アルギニン、ヒスチジン、リジン、アスパラギン酸、グルタミン酸、アスパラギン、グルタミン、ロイシン、イソロイシン、バリン、スレオニン、メチオニン、システイン、プロリン等が挙げられる。タンパク質としては、例えば、血漿由来タンパク質等が挙げられる。
Examples of inorganic salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium hydrogen carbonate, calcium nitrate, and iron nitrate. Sugars include, for example, glucose and sucrose. Amino acids include, for example, alanine, glycine, tryptophan, phenylalanine, tyrosine, arginine, histidine, lysine, aspartic acid, glutamic acid, asparagine, glutamine, leucine, isoleucine, valine, threonine, methionine, cysteine, proline, and the like. Proteins include, for example, plasma-derived proteins and the like.
血清としては、例えば、ヒト血清、ウシ血清、ウマ血清、ウシ胎児血清（Ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍ；ＦＢＳ）を用いることができる。血清代替物としては、例えば、ウシ血清アルブミン（Ｂｏｖｉｎｅ ｓｅｒｕｍ ａｌｂｕｍｉｎ；ＢＳＡ）、ノックアウト血清代替物（ＫｎｏｃｋＯｕｔ Ｓｅｒｕｍ Ｒｅｐｌａｃｅｍｅｎｔ；ＫＳＲ）、ヒト血清アルブミン（Ｈｕｍａｎ ｓｅｒｕｍ ａｌｂｕｍｉｎ；ＨＳＡまたはＨｕｍａｎ ａｌｂｕｍｉｎ，ｓｅｒｕｍ；ＨＡＳ）等が挙げられる。ビタミンとしては、例えば、ニコチンアミド、アスコルビン酸誘導体、リボフラビン、ピリドキサール、チアミン、葉酸等が挙げられる。ホルモンとしては、例えば、インスリン、エストラジオール、プロゲステロン、テストステロン、チロキシン等が挙げられる。抗生物質としては、例えば、ペニシリン、ストレプトマイシン等が挙げられる。成長因子としては、例えば、上皮増殖因子（ＥＧＦ）、線維芽細胞増殖因子－２（ＦＧＦ－２）等が挙げられる。生体組織抽出物としては、例えば、下垂体抽出物、血小板抽出物等が挙げられる。
As serum, for example, human serum, bovine serum, horse serum, and fetal bovine serum (FBS) can be used. Examples of serum substitutes include bovine serum albumin (BSA), knockout serum replacement (KSR), human serum albumin (HSA or human albumin, serum; HAS), and the like. mentioned. Examples of vitamins include nicotinamide, ascorbic acid derivatives, riboflavin, pyridoxal, thiamine, folic acid and the like. Hormones include, for example, insulin, estradiol, progesterone, testosterone, thyroxine, and the like. Examples of antibiotics include penicillin, streptomycin and the like. Growth factors include, for example, epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2) and the like. Examples of biological tissue extracts include pituitary gland extracts and platelet extracts.
培地組成物は、例えば、公知の基礎培地又はその他既存の培地に、アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種を加えることによって、製造することができる。公知の基礎培地の例として、ＤＭＥＭ（ダルベッコ改変イーグル培地）、ＭＥＭ（イーグル最小必須培地）、αＭＥＭ培地（イーグル最小必須培地α改変型）、ＧＭＥＭ（グラスゴー最小必須培地）、ＩＭＤＭ（イスコフ改変ダルベッコ培地）、Ｈａｍ’ｓ Ｆ１２（栄養混合物Ｆ－１２ハム）、ＲＰＭＩ－１６４０（ＲＰＭＩ－１６４０培地）、ＭｃＣｏｙ’ｓ ５Ａ（マッコイ５Ａ培地）、ＭＳＣ ｇｒｏｗｔｈ ｍｅｄｉｕｍ ２（Ｐｒｏｍｏｃｅｌｌ社）、Ｐｒｉｍｅ ＸＶ ＸＳＦＭ（登録商標、ＦＵＪＩＦＩＬＭ Ｉｒｖｉｎｅ Ｓｃｉｅｎｔｉｆｉｃ社）、Ｅｓｓｅｎｔｉａｌ８（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）、Ｅｓｓｅｎｔｉａｌ６（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）、及びこれらから選択される２種以上を含む混合物が挙げられる。
The medium composition includes, for example, a known basal medium or other existing medium, aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. It can be produced by adding at least one selected from the group consisting of Examples of known basal media include DMEM (Dulbecco's modified Eagle's medium), MEM (Eagle's minimal essential medium), αMEM medium (Eagle's minimal essential medium α-modified), GMEM (Glasgow's minimal essential medium), IMDM (Iscove's modified Dulbecco's medium ), Ham's F12 (nutrient mixture F-12 ham), RPMI-1640 (RPMI-1640 medium), McCoy's 5A (McCoy's 5A medium), MSC growth medium 2 (Promocell), Prime XV XSFM (registered trademark) , Fuji Film Irvine Scientific), Essential 8 (Thermo Fisher Scientific), Essential 6 (Thermo Fisher Scientific), and mixtures containing two or more selected from these.
Ｅｓｓｅｎｔｉａｌ８は、ＤＭＥＭ／Ｆ－１２、Ｌ－アスコルビン酸、セレン、トランスフェリン、ＮａＨＣＯ３、インスリン、ＦＧＦ２、及びＴＧＦβ１と、水とからなる培地である。Ｅｓｓｅｎｔｉａｌ６は、ＤＭＥＭ／Ｆ－１２、Ｌ－アスコルビン酸、セレン、トランスフェリン、ＮａＨＣＯ３、及びインスリンと、水とからなる培地である。Ｅｓｓｅｎｔｉａｌ８及びＥｓｓｅｎｔｉａｌ６は、含まれる成分が少ないために、管理が容易であり、培養のロット間のバラツキを抑えることができる。
Essential 8 is a medium consisting of DMEM/F-12, L-ascorbic acid, selenium, transferrin, NaHCO 3 , insulin, FGF2 and TGFβ1, and water. Essential 6 is a medium consisting of DMEM/F-12, L-ascorbic acid, selenium, transferrin, NaHCO 3 and insulin, and water. Since Essential 8 and Essential 6 contain few components, they are easy to manage and can suppress variation between culture lots.
培地組成物は、特に限定はなく、様々な細胞の培養に使用することができる。細胞は、動物由来の細胞であることが好ましく、哺乳動物由来の細胞であることがより好ましい。
哺乳動物として、例えば、ヒト、サル、チンパンジー、ウシ、ブタ、ウマ、ヒツジ、ヤギ、ウサギ、ラット、マウス、モルモット、イヌ、ネコ等が挙げられる。細胞として、例えば、多能性幹細胞、体性幹細胞、体細胞等が挙げられる。細胞は、多能性幹細胞であってよい。多能性幹細胞としては、例えば、ＥＳ細胞、ｉＰＳ細胞、ＥＧ細胞（Ｅｍｂｒｙｏｎｉｃ ｇｅｒｍ ｃｅｌｌ）、ＧＳ細胞（Ｇｅｒｍ－ｌｉｎｅ ｓｔｅｍ ｃｅｌｌ）が挙げられる。体性幹細胞としては、間葉系幹細胞、組織幹細胞等が挙げられる。 The medium composition is not particularly limited and can be used for culturing various cells. Cells are preferably animal-derived cells, more preferably mammalian-derived cells.
Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like. Cells include, for example, pluripotent stem cells, somatic stem cells, somatic cells, and the like. The cells may be pluripotent stem cells. Pluripotent stem cells include, for example, ES cells, iPS cells, EG cells (embryonic germ cells), and GS cells (germ-line stem cells). Somatic stem cells include mesenchymal stem cells, tissue stem cells, and the like.
哺乳動物として、例えば、ヒト、サル、チンパンジー、ウシ、ブタ、ウマ、ヒツジ、ヤギ、ウサギ、ラット、マウス、モルモット、イヌ、ネコ等が挙げられる。細胞として、例えば、多能性幹細胞、体性幹細胞、体細胞等が挙げられる。細胞は、多能性幹細胞であってよい。多能性幹細胞としては、例えば、ＥＳ細胞、ｉＰＳ細胞、ＥＧ細胞（Ｅｍｂｒｙｏｎｉｃ ｇｅｒｍ ｃｅｌｌ）、ＧＳ細胞（Ｇｅｒｍ－ｌｉｎｅ ｓｔｅｍ ｃｅｌｌ）が挙げられる。体性幹細胞としては、間葉系幹細胞、組織幹細胞等が挙げられる。 The medium composition is not particularly limited and can be used for culturing various cells. Cells are preferably animal-derived cells, more preferably mammalian-derived cells.
Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like. Cells include, for example, pluripotent stem cells, somatic stem cells, somatic cells, and the like. The cells may be pluripotent stem cells. Pluripotent stem cells include, for example, ES cells, iPS cells, EG cells (embryonic germ cells), and GS cells (germ-line stem cells). Somatic stem cells include mesenchymal stem cells, tissue stem cells, and the like.
培地組成物は、特に限定はなく、様々な培養方法の培地として、又は、その培地の調製のために使用できる。培養方法としては、細胞を固定平面に接着させて培養する二次元培養、細胞を培地中で浮遊させて培養する三次元培養が挙げられる。三次元培養を行う際には、細胞はいずれの形態であってもよく、単一細胞又は細胞塊若しくは細胞凝集体として浮遊していてもよく、マイクロキャリアのような培養担体に接着させた状態で浮遊していてもよい。本開示において、「単一細胞」とは、独立した１個の細胞を意味する。
The medium composition is not particularly limited, and can be used as a medium for various culture methods or for preparing the medium. Examples of culture methods include two-dimensional culture in which cells are adhered to a fixed surface and cultured, and three-dimensional culture in which cells are cultured while suspended in a medium. When performing three-dimensional culture, cells may be in any form, may be suspended as single cells, cell clusters, or cell aggregates, and may be adhered to a culture carrier such as a microcarrier. You can float in the In the present disclosure, "single cell" means an independent cell.
＜細胞培養物の製造方法、細胞の培養方法＞
細胞培養物の製造方法は、本開示に係る培地組成物を用いて、細胞を培養することを含む。培養には、培地組成物として、液体培地又はゲル培地を使用できる。細胞を培養する方法は特に限定されず、上述した培養方法を用いることができる。例えば、細胞培養物の製造方法は、培地組成物として液体培地を用いて、細胞を浮遊させて培養することを含む。培養する細胞も特に限定されず、上述した細胞を培養することができる。例えば、細胞培養物の製造方法は、培地組成物を用いて、多能性幹細胞を培養することを含む。これにより、多能性幹細胞と、本開示に係る培地組成物とを含む細胞培養物を得ることができる。すなわち、本開示に係る細胞培養物は、本開示に係る培地組成物と、培養済みの多能性幹細胞とを含む。 <Method for producing cell culture, method for culturing cells>
A method for producing a cell culture includes culturing cells using the medium composition according to the present disclosure. A liquid medium or a gel medium can be used as a medium composition for culture. The method for culturing the cells is not particularly limited, and the culture method described above can be used. For example, a method for producing a cell culture includes culturing cells in suspension using a liquid medium as the medium composition. Cells to be cultured are also not particularly limited, and the cells described above can be cultured. For example, a method for producing a cell culture includes culturing pluripotent stem cells using the medium composition. Thereby, a cell culture containing pluripotent stem cells and the medium composition according to the present disclosure can be obtained. That is, the cell culture according to the present disclosure includes the medium composition according to the present disclosure and cultured pluripotent stem cells.
細胞培養物の製造方法は、本開示に係る培地組成物を用いて、細胞を培養することを含む。培養には、培地組成物として、液体培地又はゲル培地を使用できる。細胞を培養する方法は特に限定されず、上述した培養方法を用いることができる。例えば、細胞培養物の製造方法は、培地組成物として液体培地を用いて、細胞を浮遊させて培養することを含む。培養する細胞も特に限定されず、上述した細胞を培養することができる。例えば、細胞培養物の製造方法は、培地組成物を用いて、多能性幹細胞を培養することを含む。これにより、多能性幹細胞と、本開示に係る培地組成物とを含む細胞培養物を得ることができる。すなわち、本開示に係る細胞培養物は、本開示に係る培地組成物と、培養済みの多能性幹細胞とを含む。 <Method for producing cell culture, method for culturing cells>
A method for producing a cell culture includes culturing cells using the medium composition according to the present disclosure. A liquid medium or a gel medium can be used as a medium composition for culture. The method for culturing the cells is not particularly limited, and the culture method described above can be used. For example, a method for producing a cell culture includes culturing cells in suspension using a liquid medium as the medium composition. Cells to be cultured are also not particularly limited, and the cells described above can be cultured. For example, a method for producing a cell culture includes culturing pluripotent stem cells using the medium composition. Thereby, a cell culture containing pluripotent stem cells and the medium composition according to the present disclosure can be obtained. That is, the cell culture according to the present disclosure includes the medium composition according to the present disclosure and cultured pluripotent stem cells.
培養温度は、培養方法に関わらず、３６．０～３８．０℃、又は３６．５～３７．５℃であってよい。培養雰囲気は、ＣＯ２インキュベーター等を使用して調整できる。培養雰囲気は、１～１０％、又は３～７％のＣＯ２雰囲気であってよい。
The culture temperature may be 36.0-38.0°C, or 36.5-37.5°C, regardless of the culture method. The culture atmosphere can be adjusted using a CO2 incubator or the like. The culture atmosphere may be a 1-10%, or 3-7% CO 2 atmosphere.
細胞培養物の製造方法は、任意の工程を含んでよい。任意の工程として、例えば、凍結された細胞を解凍すること、細胞を洗浄すること、細胞を播種すること、培地又は培地に含まれる少なくとも１種の成分を細胞培養物に追加すること、細胞培養物に含まれる少なくとも一部の培地を交換すること、細胞培養物を静置すること、所定の培養期間後に細胞を、フィルタ等を用いて培地組成物と分離して回収すること、回収された細胞を保存用組成物と組み合わせて保存すること等が挙げられる。保存用組成物としては、他の公知の保存用培地、凍結保存用組成物等が挙げられる。
A method for producing a cell culture may include any steps. Optional steps include, for example, thawing frozen cells, washing cells, seeding cells, adding medium or at least one component contained in medium to cell culture, cell culture exchanging at least part of the medium contained in the product, allowing the cell culture to stand, separating the cells from the medium composition using a filter or the like after a predetermined culture period, and collecting the collected Examples include preserving the cells in combination with a preserving composition. Preservation compositions include other known preservation media, cryopreservation compositions, and the like.
細胞の培養方法は、本開示に係る培地組成物を用いて、細胞を培養することを含む。上記の細胞培養物の製造方法に関する説明を、細胞の培養方法にも適用できる。
A method for culturing cells includes culturing cells using the medium composition according to the present disclosure. The above description of the cell culture production method can also be applied to the cell culture method.
＜細胞培養物＞
細胞培養物の製造方法又は細胞の培養方法によって、細胞培養物が得られる。細胞培養物は、本開示に係る培地組成物と、細胞とを含む。培養後の細胞培養物からは、増殖後の細胞を回収することができる。 <Cell culture>
A cell culture is obtained by a method for producing a cell culture or a method for culturing cells. A cell culture comprises a medium composition according to the present disclosure and cells. Proliferated cells can be recovered from the cultured cell culture.
細胞培養物の製造方法又は細胞の培養方法によって、細胞培養物が得られる。細胞培養物は、本開示に係る培地組成物と、細胞とを含む。培養後の細胞培養物からは、増殖後の細胞を回収することができる。 <Cell culture>
A cell culture is obtained by a method for producing a cell culture or a method for culturing cells. A cell culture comprises a medium composition according to the present disclosure and cells. Proliferated cells can be recovered from the cultured cell culture.
＜細胞懸濁物＞
細胞懸濁物は、本開示に係る培地組成物と、多能性幹細胞とを含有する。多能性幹細胞は、本開示に係る培地組成物を用いて培養された多能性幹細胞であってもよい。本細胞懸濁物をそのまま多能性幹細胞の培養に用いることで、従来よりも高い培養効率で多能性幹細胞の培養を開始することができ、又は培養を継続することができる。 <Cell suspension>
A cell suspension contains a medium composition according to the present disclosure and pluripotent stem cells. Pluripotent stem cells may be pluripotent stem cells cultured using the medium composition of the present disclosure. By using the present cell suspension as it is for culturing pluripotent stem cells, culturing of pluripotent stem cells can be started or continued with higher culture efficiency than before.
細胞懸濁物は、本開示に係る培地組成物と、多能性幹細胞とを含有する。多能性幹細胞は、本開示に係る培地組成物を用いて培養された多能性幹細胞であってもよい。本細胞懸濁物をそのまま多能性幹細胞の培養に用いることで、従来よりも高い培養効率で多能性幹細胞の培養を開始することができ、又は培養を継続することができる。 <Cell suspension>
A cell suspension contains a medium composition according to the present disclosure and pluripotent stem cells. Pluripotent stem cells may be pluripotent stem cells cultured using the medium composition of the present disclosure. By using the present cell suspension as it is for culturing pluripotent stem cells, culturing of pluripotent stem cells can be started or continued with higher culture efficiency than before.
＜培養により得られる細胞＞
本開示に係る培地組成物を用いる培養により得られる細胞は、未分化能を有することができる。細胞の未分化能は、当業界で既知の確認方法によって確認することができる。未分化能を確認するための方法としては、例えば、遺伝子、タンパク質、糖脂質等の既知の因子の発現量、存在量等を単独で又は組み合わせて測定する方法が挙げられる。このような因子をマーカーと称することがあり、またマーカーとしての遺伝子及びタンパク質を、それぞれマーカー遺伝子及びマーカータンパク質と称することがある。 <Cells obtained by culture>
Cells obtained by culturing using the medium composition according to the present disclosure can have undifferentiation potential. The undifferentiated potential of cells can be confirmed by confirmation methods known in the art. Methods for confirming undifferentiated potency include, for example, methods for measuring the expression levels, abundance, and the like of known factors such as genes, proteins, and glycolipids, singly or in combination. Such factors are sometimes referred to as markers, and the genes and proteins used as markers are sometimes referred to as marker genes and marker proteins, respectively.
本開示に係る培地組成物を用いる培養により得られる細胞は、未分化能を有することができる。細胞の未分化能は、当業界で既知の確認方法によって確認することができる。未分化能を確認するための方法としては、例えば、遺伝子、タンパク質、糖脂質等の既知の因子の発現量、存在量等を単独で又は組み合わせて測定する方法が挙げられる。このような因子をマーカーと称することがあり、またマーカーとしての遺伝子及びタンパク質を、それぞれマーカー遺伝子及びマーカータンパク質と称することがある。 <Cells obtained by culture>
Cells obtained by culturing using the medium composition according to the present disclosure can have undifferentiation potential. The undifferentiated potential of cells can be confirmed by confirmation methods known in the art. Methods for confirming undifferentiated potency include, for example, methods for measuring the expression levels, abundance, and the like of known factors such as genes, proteins, and glycolipids, singly or in combination. Such factors are sometimes referred to as markers, and the genes and proteins used as markers are sometimes referred to as marker genes and marker proteins, respectively.
マーカーとしては、Ｎａｎｏｇ、Ｓｏｘ２、Ｏｃｔ－４、Ｋｌｆ４、Ｌｉｎ２８、ＴＲＡ－１－６０、ＴＲＡ－１－８１、ＴＲＡ－２－４９／６Ｅ、ＳＳＥＡ－１、ＳＳＥＡ－３、ＳＳＥＡ－４、ｃ－Ｍｙｃ等が挙げられる。これらのマーカーを単独で又は２つ以上を組み合わせて、細胞の未分化能を評価に使用することができる。マーカーを用いた細胞の未分化能の具体的な確認方法としては、定量的ＰＣＲ等によりマーカー遺伝子の発現量を測定する方法、フローサイトメトリー等の免疫測定法により表面抗原等のマーカーの存在有無又は存在量を確認又は測定する方法などが挙げられる。未分化能を有する細胞は、細胞の種類、由来等により、例えば、未分化能を示さない既知の細胞と比較して、マーカーとしてＮａｎｏｇ、Ｓｏｘ２、Ｏｃｔ－４、Ｋｌｆ４、Ｌｉｎ２８、ＴＲＡ－１－６０、ＴＲＡ－１－８１、ＴＲＡ－２－４９／６Ｅ、ＳＳＥＡ－１、ＳＳＥＡ－３、ＳＳＥＡ－４、ｃ－Ｍｙｃ等の１つ又は２つ以上、例えば、Ｎａｎｏｇ、Ｓｏｘ２、Ｏｃｔ－４、ＳＳＥＡ－４等の１つ又は２つ以上の存在量が増加する傾向を示す場合がある。このため、未分化能を有する細胞は、細胞の種類、由来等により、例えば、未分化能を示さない既知の細胞と比較して、高Ｎａｎｏｇ量、高Ｓｏｘ２量、高Ｏｃｔ－４量、高Ｋｌｆ４量、高Ｌｉｎ２８量、高ＴＲＡ－１－６０量、高ＴＲＡ－１－８１量、高ＴＲＡ－２－４９／６Ｅ量、高ＳＳＥＡ－１量、高ＳＳＥＡ－３量、高ＳＳＥＡ－４量、高ｃ－Ｍｙｃ量等の１つ又は２つ以上、例えば、高Ｎａｎｏｇ量、高Ｓｏｘ２量、高Ｏｃｔ－４量、高ＳＳＥＡ－４量の１つ又は２つ以上の特徴を示す細胞として特定することができる。
Markers include Nanog, Sox2, Oct-4, Klf4, Lin28, TRA-1-60, TRA-1-81, TRA-2-49/6E, SSEA-1, SSEA-3, SSEA-4, c- and Myc. These markers can be used alone or in combination of two or more to evaluate the undifferentiated potential of cells. Specific methods for confirming the undifferentiated potential of cells using markers include a method of measuring the expression level of marker genes by quantitative PCR, etc., and the presence or absence of markers such as surface antigens by immunoassays such as flow cytometry. Alternatively, a method of confirming or measuring the abundance can be used. Cells with undifferentiated potential can be compared with known cells that do not exhibit undifferentiated potential depending on the cell type, origin, etc., and can be compared with markers such as Nanog, Sox2, Oct-4, Klf4, Lin28, TRA-1- 60, TRA-1-81, TRA-2-49/6E, SSEA-1, SSEA-3, SSEA-4, c-Myc, etc., for example Nanog, Sox2, Oct-4, There may be a trend towards increased abundance of one or more, such as SSEA-4. For this reason, cells with undifferentiated potency are, for example, compared to known cells that do not show undifferentiated potency, due to the cell type, origin, etc., having a high Nanog amount, a high Sox2 amount, a high Oct-4 amount, a high Klf4 amount, high Lin28 amount, high TRA-1-60 amount, high TRA-1-81 amount, high TRA-2-49/6E amount, high SSEA-1 amount, high SSEA-3 amount, high SSEA-4 amount , high c-Myc amount, etc., for example, high Nanog amount, high Sox2 amount, high Oct-4 amount, high SSEA-4 amount. can do.
実施形態の例を以下に列挙する。本発明は以下の例に限定されない。
（１） アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種を含有する、培地組成物。
（２） 前記アミノアルキルスルホン酸を含有する、上記（１）に記載の培地組成物。
（３） 前記アミノアルキルスルホン酸が、２－アミノエチルスルホン酸を含む、上記（２）に記載の培地組成物。
（４） 前記カルボン酸鉄塩を含有する、上記（１）～（３）のいずれか１項に記載の培地組成物。
（５） 前記カルボン酸鉄塩が、クエン酸鉄アンモニウムを含む、上記（４）に記載の培地組成物。
（６） 前記アミノアルキルスルホン酸と前記カルボン酸鉄塩とを含有する、上記（１）に記載の培地組成物。
（７） 前記アミノアルキルスルホン酸及び前記カルボン酸鉄塩がそれぞれ、２－アミノエチルスルホン酸及びクエン酸鉄アンモニウムを含有する、上記（６）に記載の培地組成物。
（８） 前記ジメチルオキサリルグリシンを含有する、上記（１）～（７）のいずれか１項に記載の培地組成物。
（９） 前記４－フェニル酪酸又は前記４－フェニル酪酸塩を含有する、上記（１）～（８）のいずれか１項に記載の培地組成物。
（１０） 細胞の浮遊培養に用いられる、上記（１）～（９）のいずれか１項に記載の培地組成物。又は、（１０’）上記（１）～（９）のいずれか１項に記載の培地組成物の、細胞の浮遊培養のための使用。
（１１） 多能性幹細胞の培養に用いられる、上記（１）～（１０）のいずれか１項に記載の培地組成物。又は、（１１’）上記（１）～（１０）のいずれか１項に記載の培地組成物の、多能性幹細胞の培養のための使用。前記培養は、浮遊培養であってよい。
（１２） 上記（１）～（１１）のいずれか１項に記載の培地組成物を用いて、細胞を培養することを含む、細胞培養物の製造方法。
（１３） 前記細胞を、浮遊させて培養する、上記（１２）に記載の細胞培養物の製造方法。
（１４） 前記細胞が、多能性幹細胞を含む、上記（１２）又は（１３）に記載の細胞培養物の製造方法。
（１５） 上記（１）～（１１）のいずれか１項に記載の培地組成物と、多能性幹細胞とを含有する、細胞懸濁物。 Examples of embodiments are listed below. The invention is not limited to the following examples.
(1) A medium containing at least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. Composition.
(2) The medium composition according to (1) above, which contains the aminoalkylsulfonic acid.
(3) The medium composition according to (2) above, wherein the aminoalkylsulfonic acid comprises 2-aminoethylsulfonic acid.
(4) The medium composition according to any one of (1) to (3) above, which contains the iron carboxylate.
(5) The medium composition according to (4) above, wherein the iron carboxylate contains ferric ammonium citrate.
(6) The medium composition according to (1) above, which contains the aminoalkylsulfonic acid and the iron carboxylate.
(7) The medium composition according to (6) above, wherein the aminoalkylsulfonic acid and the carboxylic acid iron salt contain 2-aminoethylsulfonic acid and ferric ammonium citrate, respectively.
(8) The medium composition according to any one of (1) to (7) above, which contains the dimethyloxalylglycine.
(9) The medium composition according to any one of (1) to (8) above, which contains the 4-phenylbutyric acid or the 4-phenylbutyric acid salt.
(10) The medium composition according to any one of (1) to (9) above, which is used for suspension culture of cells. Alternatively, (10') use of the medium composition according to any one of (1) to (9) above for suspension culture of cells.
(11) The medium composition according to any one of (1) to (10) above, which is used for culturing pluripotent stem cells. Alternatively, (11′) use of the medium composition according to any one of (1) to (10) above for culturing pluripotent stem cells. The culture may be suspension culture.
(12) A method for producing a cell culture, comprising culturing cells using the medium composition according to any one of (1) to (11) above.
(13) The method for producing a cell culture according to (12) above, wherein the cells are cultured in suspension.
(14) The method for producing a cell culture according to (12) or (13) above, wherein the cells comprise pluripotent stem cells.
(15) A cell suspension containing the medium composition according to any one of (1) to (11) above and pluripotent stem cells.
（１） アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種を含有する、培地組成物。
（２） 前記アミノアルキルスルホン酸を含有する、上記（１）に記載の培地組成物。
（３） 前記アミノアルキルスルホン酸が、２－アミノエチルスルホン酸を含む、上記（２）に記載の培地組成物。
（４） 前記カルボン酸鉄塩を含有する、上記（１）～（３）のいずれか１項に記載の培地組成物。
（５） 前記カルボン酸鉄塩が、クエン酸鉄アンモニウムを含む、上記（４）に記載の培地組成物。
（６） 前記アミノアルキルスルホン酸と前記カルボン酸鉄塩とを含有する、上記（１）に記載の培地組成物。
（７） 前記アミノアルキルスルホン酸及び前記カルボン酸鉄塩がそれぞれ、２－アミノエチルスルホン酸及びクエン酸鉄アンモニウムを含有する、上記（６）に記載の培地組成物。
（８） 前記ジメチルオキサリルグリシンを含有する、上記（１）～（７）のいずれか１項に記載の培地組成物。
（９） 前記４－フェニル酪酸又は前記４－フェニル酪酸塩を含有する、上記（１）～（８）のいずれか１項に記載の培地組成物。
（１０） 細胞の浮遊培養に用いられる、上記（１）～（９）のいずれか１項に記載の培地組成物。又は、（１０’）上記（１）～（９）のいずれか１項に記載の培地組成物の、細胞の浮遊培養のための使用。
（１１） 多能性幹細胞の培養に用いられる、上記（１）～（１０）のいずれか１項に記載の培地組成物。又は、（１１’）上記（１）～（１０）のいずれか１項に記載の培地組成物の、多能性幹細胞の培養のための使用。前記培養は、浮遊培養であってよい。
（１２） 上記（１）～（１１）のいずれか１項に記載の培地組成物を用いて、細胞を培養することを含む、細胞培養物の製造方法。
（１３） 前記細胞を、浮遊させて培養する、上記（１２）に記載の細胞培養物の製造方法。
（１４） 前記細胞が、多能性幹細胞を含む、上記（１２）又は（１３）に記載の細胞培養物の製造方法。
（１５） 上記（１）～（１１）のいずれか１項に記載の培地組成物と、多能性幹細胞とを含有する、細胞懸濁物。 Examples of embodiments are listed below. The invention is not limited to the following examples.
(1) A medium containing at least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate. Composition.
(2) The medium composition according to (1) above, which contains the aminoalkylsulfonic acid.
(3) The medium composition according to (2) above, wherein the aminoalkylsulfonic acid comprises 2-aminoethylsulfonic acid.
(4) The medium composition according to any one of (1) to (3) above, which contains the iron carboxylate.
(5) The medium composition according to (4) above, wherein the iron carboxylate contains ferric ammonium citrate.
(6) The medium composition according to (1) above, which contains the aminoalkylsulfonic acid and the iron carboxylate.
(7) The medium composition according to (6) above, wherein the aminoalkylsulfonic acid and the carboxylic acid iron salt contain 2-aminoethylsulfonic acid and ferric ammonium citrate, respectively.
(8) The medium composition according to any one of (1) to (7) above, which contains the dimethyloxalylglycine.
(9) The medium composition according to any one of (1) to (8) above, which contains the 4-phenylbutyric acid or the 4-phenylbutyric acid salt.
(10) The medium composition according to any one of (1) to (9) above, which is used for suspension culture of cells. Alternatively, (10') use of the medium composition according to any one of (1) to (9) above for suspension culture of cells.
(11) The medium composition according to any one of (1) to (10) above, which is used for culturing pluripotent stem cells. Alternatively, (11′) use of the medium composition according to any one of (1) to (10) above for culturing pluripotent stem cells. The culture may be suspension culture.
(12) A method for producing a cell culture, comprising culturing cells using the medium composition according to any one of (1) to (11) above.
(13) The method for producing a cell culture according to (12) above, wherein the cells are cultured in suspension.
(14) The method for producing a cell culture according to (12) or (13) above, wherein the cells comprise pluripotent stem cells.
(15) A cell suspension containing the medium composition according to any one of (1) to (11) above and pluripotent stem cells.
本発明の実施形態について実施例により具体的に説明する。本発明の実施形態は以下の実施例に限定されない。
The embodiments of the present invention will be specifically described with examples. Embodiments of the invention are not limited to the following examples.
［実施例１］
細胞として、国立大学法人 京都大学より譲渡を受けたヒトｉＰＳ細胞（２０１Ｂ７株）を使用した。 [Example 1]
Human iPS cells (strain 201B7) obtained from National University Corporation Kyoto University were used as cells.
細胞として、国立大学法人 京都大学より譲渡を受けたヒトｉＰＳ細胞（２０１Ｂ７株）を使用した。 [Example 1]
Human iPS cells (strain 201B7) obtained from National University Corporation Kyoto University were used as cells.
（細胞培養液（液体培地）の調製）
Ｅｓｓｅｎｔｉａｌ６（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）５００ｍＬに、ｂＦＧＦ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）５０μｇ、及び２－アミノエチルスルホン酸（タウリン、富士フイルム和光純薬株式会社）６２．５ｍｇを添加し、細胞培養液１を調製した。なお、ｂＦＧＦの添加量は１００ｎｇ／ｍＬであり、２－アミノエチルスルホン酸の添加量は１２５μｇ／ｍＬであった。 (Preparation of cell culture medium (liquid medium))
50 μg of bFGF (R&D Systems) and 62.5 mg of 2-aminoethylsulfonic acid (taurine, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to 500 mL of Essential 6 (Thermo Fisher Scientific) to prepare cell culture solution 1. . The amount of bFGF added was 100 ng/mL, and the amount of 2-aminoethylsulfonic acid added was 125 μg/mL.
Ｅｓｓｅｎｔｉａｌ６（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）５００ｍＬに、ｂＦＧＦ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）５０μｇ、及び２－アミノエチルスルホン酸（タウリン、富士フイルム和光純薬株式会社）６２．５ｍｇを添加し、細胞培養液１を調製した。なお、ｂＦＧＦの添加量は１００ｎｇ／ｍＬであり、２－アミノエチルスルホン酸の添加量は１２５μｇ／ｍＬであった。 (Preparation of cell culture medium (liquid medium))
50 μg of bFGF (R&D Systems) and 62.5 mg of 2-aminoethylsulfonic acid (taurine, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to 500 mL of Essential 6 (Thermo Fisher Scientific) to prepare cell culture solution 1. . The amount of bFGF added was 100 ng/mL, and the amount of 2-aminoethylsulfonic acid added was 125 μg/mL.
（２０１Ｂ７の浮遊培養）
低接着コーティングされた６ｃｍペトリディッシュ（ＡＧＣテクノグラス株式会社）に、細胞培養液１を６ｍＬ滴下し、次いで２０１Ｂ７を２．５×１０５ｃｅｌｌｓ播種した。ペトリディッシュを３７℃、ＣＯ２濃度５％のインキュベーター内に静置し、浮遊培養を実施した。培養は１５日間継続させ、培養３日目、７日目、１０日目、及び１５日目に、生細胞数を計測した。 (Suspension culture of 201B7)
6 mL of the cell culture medium 1 was dropped on a low-adhesion coated 6 cm Petri dish (AGC Techno Glass Co., Ltd.), and then 2.5×10 5 cells of 201B7 were seeded. The Petri dish was placed in an incubator at 37° C. and a CO 2 concentration of 5% to carry out suspension culture. The culture was continued for 15 days, and the number of viable cells was counted on the 3rd, 7th, 10th and 15th days of the culture.
低接着コーティングされた６ｃｍペトリディッシュ（ＡＧＣテクノグラス株式会社）に、細胞培養液１を６ｍＬ滴下し、次いで２０１Ｂ７を２．５×１０５ｃｅｌｌｓ播種した。ペトリディッシュを３７℃、ＣＯ２濃度５％のインキュベーター内に静置し、浮遊培養を実施した。培養は１５日間継続させ、培養３日目、７日目、１０日目、及び１５日目に、生細胞数を計測した。 (Suspension culture of 201B7)
6 mL of the cell culture medium 1 was dropped on a low-adhesion coated 6 cm Petri dish (AGC Techno Glass Co., Ltd.), and then 2.5×10 5 cells of 201B7 were seeded. The Petri dish was placed in an incubator at 37° C. and a CO 2 concentration of 5% to carry out suspension culture. The culture was continued for 15 days, and the number of viable cells was counted on the 3rd, 7th, 10th and 15th days of the culture.
（生細胞数の計測）
細胞計測装置ＮＣ－２００（ＮｕｃｌｅｏＣｏｕｎｔｅｒ（登録商標）、ＣｈｅｍｏＭｅｔｅｃ社）を用いて、プロトコールに従い生細胞数の計測を行った。酵素処理後の細胞懸濁液を一部採取し、細胞計測装置専用のＶｉａ１カセットで細胞懸濁液を吸引した。Ｖｉａ１カセットをＮＣ－２００にセットして測定を実施した。 (Measurement of viable cell count)
Using a cell counter NC-200 (NucleoCounter (registered trademark), ChemoMetec), the number of viable cells was counted according to the protocol. A portion of the cell suspension after the enzyme treatment was collected, and the cell suspension was aspirated with a Via1 cassette dedicated to the cell measuring device. Measurement was carried out by setting the Via1 cassette in the NC-200.
細胞計測装置ＮＣ－２００（ＮｕｃｌｅｏＣｏｕｎｔｅｒ（登録商標）、ＣｈｅｍｏＭｅｔｅｃ社）を用いて、プロトコールに従い生細胞数の計測を行った。酵素処理後の細胞懸濁液を一部採取し、細胞計測装置専用のＶｉａ１カセットで細胞懸濁液を吸引した。Ｖｉａ１カセットをＮＣ－２００にセットして測定を実施した。 (Measurement of viable cell count)
Using a cell counter NC-200 (NucleoCounter (registered trademark), ChemoMetec), the number of viable cells was counted according to the protocol. A portion of the cell suspension after the enzyme treatment was collected, and the cell suspension was aspirated with a Via1 cassette dedicated to the cell measuring device. Measurement was carried out by setting the Via1 cassette in the NC-200.
（ＰＤＬの算出）
計測により得られた生細胞数から、下記式を用いて細胞集団倍加数（Ｐｏｐｕｌａｔｉｏｎ ｄｏｕｂｌｉｎｇ ｌｅｖｅｌ；ＰＤＬ。Tissue Culture Methods and Applications, (Academic Press, New York), L. Hayflick, pp.220-223, (1973)）を算出した。式中、細胞の収量をＵＣＹ、培養のために播種した細胞の数をｌ、播種された細胞集団の当初の分裂回数をＸとした。
ＰＤＬ ＝ ３．３２ （ｌｏｇ ＵＣＹ － ｌｏｇ ｌ） ＋ Ｘ
(Calculation of PDL)
From the number of living cells obtained by measurement, the cell population doubling level (PDL. Tissue Culture Methods and Applications, (Academic Press, New York), L. Hayflick, pp.220-223, (1973)) was calculated. where UCY was the cell yield, l was the number of cells seeded for culture, and X was the initial number of divisions of the seeded cell population.
PDL = 3.32 (log UCY - log l) + X
計測により得られた生細胞数から、下記式を用いて細胞集団倍加数（Ｐｏｐｕｌａｔｉｏｎ ｄｏｕｂｌｉｎｇ ｌｅｖｅｌ；ＰＤＬ。Tissue Culture Methods and Applications, (Academic Press, New York), L. Hayflick, pp.220-223, (1973)）を算出した。式中、細胞の収量をＵＣＹ、培養のために播種した細胞の数をｌ、播種された細胞集団の当初の分裂回数をＸとした。
ＰＤＬ ＝ ３．３２ （ｌｏｇ ＵＣＹ － ｌｏｇ ｌ） ＋ Ｘ
(Calculation of PDL)
From the number of living cells obtained by measurement, the cell population doubling level (PDL. Tissue Culture Methods and Applications, (Academic Press, New York), L. Hayflick, pp.220-223, (1973)) was calculated. where UCY was the cell yield, l was the number of cells seeded for culture, and X was the initial number of divisions of the seeded cell population.
PDL = 3.32 (log UCY - log l) + X
［実施例２］
実施例１に記載のとおり調製した細胞培養液１ ６ｍＬに、クエン酸鉄アンモニウム（富士フイルム和光純薬株式会社）１．５μｇを添加し、細胞培養液２を調製した。なお、ｂＦＧＦの添加量は１００ｎｇ／ｍＬであり、２－アミノエチルスルホン酸の添加量は１２５μｇ／ｍＬであり、クエン酸鉄アンモニウムの添加量は０．２５μｇ／ｍＬであった。
続いて、実施例１に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。 [Example 2]
To 6 mL of cell culture medium 1 prepared as described in Example 1, 1.5 μg of ferric ammonium citrate (Fuji Film Wako Pure Chemical Industries, Ltd.) was added to prepare cell culture medium 2. The amount of bFGF added was 100 ng/mL, the amount of 2-aminoethylsulfonic acid added was 125 μg/mL, and the amount of ferric ammonium citrate added was 0.25 μg/mL.
Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells.
実施例１に記載のとおり調製した細胞培養液１ ６ｍＬに、クエン酸鉄アンモニウム（富士フイルム和光純薬株式会社）１．５μｇを添加し、細胞培養液２を調製した。なお、ｂＦＧＦの添加量は１００ｎｇ／ｍＬであり、２－アミノエチルスルホン酸の添加量は１２５μｇ／ｍＬであり、クエン酸鉄アンモニウムの添加量は０．２５μｇ／ｍＬであった。
続いて、実施例１に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。 [Example 2]
To 6 mL of cell culture medium 1 prepared as described in Example 1, 1.5 μg of ferric ammonium citrate (Fuji Film Wako Pure Chemical Industries, Ltd.) was added to prepare cell culture medium 2. The amount of bFGF added was 100 ng/mL, the amount of 2-aminoethylsulfonic acid added was 125 μg/mL, and the amount of ferric ammonium citrate added was 0.25 μg/mL.
Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells.
［比較例１］
Ｅｓｓｅｎｔｉａｌ６（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）６ｍＬに、ｂＦＧＦ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）６００ｎｇを添加し、細胞培養液を調製した。
続いて、実施例１に記載の手順にと同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。 [Comparative Example 1]
600 ng of bFGF (R&D Systems) was added to 6 mL of Essential 6 (Thermo Fisher Scientific) to prepare a cell culture solution.
Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells.
Ｅｓｓｅｎｔｉａｌ６（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）６ｍＬに、ｂＦＧＦ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）６００ｎｇを添加し、細胞培養液を調製した。
続いて、実施例１に記載の手順にと同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。 [Comparative Example 1]
600 ng of bFGF (R&D Systems) was added to 6 mL of Essential 6 (Thermo Fisher Scientific) to prepare a cell culture solution.
Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells.
（ＰＤＬの比較）
表１に、実施例１及び２と比較例１におけるＰＤＬ算出結果を示す。 (PDL comparison)
Table 1 shows the PDL calculation results in Examples 1 and 2 and Comparative Example 1.
表１に、実施例１及び２と比較例１におけるＰＤＬ算出結果を示す。 (PDL comparison)
Table 1 shows the PDL calculation results in Examples 1 and 2 and Comparative Example 1.
［実施例３］
Ｅｓｓｅｎｔｉａｌ６（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）６ｍＬに、ｂＦＧＦ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）６００ｎｇ、クエン酸鉄アンモニウム（富士フイルム和光純薬株式会社）１．５μｇを添加し、細胞培養液３を調製した。クエン酸鉄アンモニウムの添加量は、０．２５μｇ／ｍＬであった。続いて、実施例１に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。ＰＤＬは、培養４日目において３．３８であった。 [Example 3]
To 6 mL of Essential 6 (Thermo Fisher Scientific), 600 ng of bFGF (R&D Systems) and 1.5 μg of ferric ammonium citrate (Fuji Film Wako Pure Chemical Industries, Ltd.) were added to prepare a cell culture solution 3. The amount of ferric ammonium citrate added was 0.25 μg/mL. Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells. PDL was 3.38 on day 4 of culture.
Ｅｓｓｅｎｔｉａｌ６（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）６ｍＬに、ｂＦＧＦ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）６００ｎｇ、クエン酸鉄アンモニウム（富士フイルム和光純薬株式会社）１．５μｇを添加し、細胞培養液３を調製した。クエン酸鉄アンモニウムの添加量は、０．２５μｇ／ｍＬであった。続いて、実施例１に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。ＰＤＬは、培養４日目において３．３８であった。 [Example 3]
To 6 mL of Essential 6 (Thermo Fisher Scientific), 600 ng of bFGF (R&D Systems) and 1.5 μg of ferric ammonium citrate (Fuji Film Wako Pure Chemical Industries, Ltd.) were added to prepare a cell culture solution 3. The amount of ferric ammonium citrate added was 0.25 μg/mL. Subsequently, suspension culture was performed in the same manner as in Example 1, and PDL was calculated from the results of counting the number of viable cells. PDL was 3.38 on day 4 of culture.
［比較例２］
比較例１に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果からＰＤＬを算出した。ＰＤＬは、培養４日目において２．７８であった。 [Comparative Example 2]
Suspension culture was performed in the same manner as in Comparative Example 1, and PDL was calculated from the results of counting the number of viable cells. PDL was 2.78 on day 4 of culture.
比較例１に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果からＰＤＬを算出した。ＰＤＬは、培養４日目において２．７８であった。 [Comparative Example 2]
Suspension culture was performed in the same manner as in Comparative Example 1, and PDL was calculated from the results of counting the number of viable cells. PDL was 2.78 on day 4 of culture.
実施例１～３と比較例１及び２のＰＤＬの比較から、２－アミノエチルスルホン酸、クエン酸鉄アンモニウム、又はこれらの両方の添加は、ｉＰＳ細胞の増殖性向上に有効であるとの結論を得た。
From the comparison of PDL in Examples 1-3 and Comparative Examples 1 and 2, the conclusion that the addition of 2-aminoethylsulfonic acid, ferric ammonium citrate, or both of these is effective in improving the proliferation of iPS cells. got
［実施例４］
（細胞培養液（液体培地）の調製）
ＤＭＥＭ／Ｆ１２をベースとする基準培地１．５ｍＬに、ジメチルオキサリルグリシン（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）５ｎＭを添加し、細胞培養液４を調製した。ジメチルオキサリルグリシンの添加量は、０．８８ｎｇ／ｍＬであった。 [Example 4]
(Preparation of cell culture medium (liquid medium))
Cell culture medium 4 was prepared by adding 5 nM of dimethyloxalylglycine (R&D systems) to 1.5 mL of DMEM/F12-based reference medium. The amount of dimethyloxalylglycine added was 0.88 ng/mL.
（細胞培養液（液体培地）の調製）
ＤＭＥＭ／Ｆ１２をベースとする基準培地１．５ｍＬに、ジメチルオキサリルグリシン（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）５ｎＭを添加し、細胞培養液４を調製した。ジメチルオキサリルグリシンの添加量は、０．８８ｎｇ／ｍＬであった。 [Example 4]
(Preparation of cell culture medium (liquid medium))
Cell culture medium 4 was prepared by adding 5 nM of dimethyloxalylglycine (R&D systems) to 1.5 mL of DMEM/F12-based reference medium. The amount of dimethyloxalylglycine added was 0.88 ng/mL.
（２０１Ｂ７の浮遊培養）
低接着コーティングされた６ウェルプレート（ＡＧＣテクノグラス株式会社）１ウェルに、細胞培養液４を１．５ｍＬ滴下し、次いで２０１Ｂ７を１．３×１０５ｃｅｌｌｓ播種した。６ウェルプレートを３７℃、ＣＯ２濃度５％のインキュベーター内に設置したオービタルシェイカーに載せ、浮遊培養を実施した。培養を７日間継続し、実施例１に記載の手順と同様に、生細胞数の計測結果からＰＤＬを算出した。ＰＤＬは、培養７日目において２．５１であった。 (Suspension culture of 201B7)
1.5 mL of the cell culture medium 4 was dropped on one well of a low-adhesion coated 6-well plate (AGC Techno Glass Co., Ltd.), and then 1.3×10 5 cells of 201B7 were seeded. The 6-well plate was placed on an orbital shaker placed in an incubator at 37° C. and 5% CO 2 concentration to perform suspension culture. Cultivation was continued for 7 days, and PDL was calculated from the results of viable cell count in the same manner as in Example 1. PDL was 2.51 on day 7 of culture.
低接着コーティングされた６ウェルプレート（ＡＧＣテクノグラス株式会社）１ウェルに、細胞培養液４を１．５ｍＬ滴下し、次いで２０１Ｂ７を１．３×１０５ｃｅｌｌｓ播種した。６ウェルプレートを３７℃、ＣＯ２濃度５％のインキュベーター内に設置したオービタルシェイカーに載せ、浮遊培養を実施した。培養を７日間継続し、実施例１に記載の手順と同様に、生細胞数の計測結果からＰＤＬを算出した。ＰＤＬは、培養７日目において２．５１であった。 (Suspension culture of 201B7)
1.5 mL of the cell culture medium 4 was dropped on one well of a low-adhesion coated 6-well plate (AGC Techno Glass Co., Ltd.), and then 1.3×10 5 cells of 201B7 were seeded. The 6-well plate was placed on an orbital shaker placed in an incubator at 37° C. and 5% CO 2 concentration to perform suspension culture. Cultivation was continued for 7 days, and PDL was calculated from the results of viable cell count in the same manner as in Example 1. PDL was 2.51 on day 7 of culture.
［実施例５］
ＤＭＥＭ／Ｆ１２をベースとする基準培地１．５ｍＬに、４－フェニル酪酸（Ｓｉｇｍａ）１０μＭを添加し、細胞培養液５を調製した。４－フェニル酪酸の添加量は、１．６μｇ／ｍＬであった。続いて、実施例４に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。ＰＤＬは、培養７日目において２．２９であった。 [Example 5]
Cell culture medium 5 was prepared by adding 10 μM 4-phenylbutyric acid (Sigma) to 1.5 mL of DMEM/F12-based reference medium. The amount of 4-phenylbutyric acid added was 1.6 μg/mL. Subsequently, suspension culture was performed in the same manner as in Example 4, and PDL was calculated from the results of counting the number of viable cells. PDL was 2.29 on day 7 of culture.
ＤＭＥＭ／Ｆ１２をベースとする基準培地１．５ｍＬに、４－フェニル酪酸（Ｓｉｇｍａ）１０μＭを添加し、細胞培養液５を調製した。４－フェニル酪酸の添加量は、１．６μｇ／ｍＬであった。続いて、実施例４に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。ＰＤＬは、培養７日目において２．２９であった。 [Example 5]
Cell culture medium 5 was prepared by adding 10 μM 4-phenylbutyric acid (Sigma) to 1.5 mL of DMEM/F12-based reference medium. The amount of 4-phenylbutyric acid added was 1.6 μg/mL. Subsequently, suspension culture was performed in the same manner as in Example 4, and PDL was calculated from the results of counting the number of viable cells. PDL was 2.29 on day 7 of culture.
［比較例３］
ＤＭＥＭ／Ｆ１２をベースとする基準培地１．５ｍＬを細胞培養液として使用する以外は実施例４に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。ＰＤＬは、培養７日目において１．６９であった。 [Comparative Example 3]
Suspension culture was performed in the same manner as in Example 4, except that 1.5 mL of a DMEM/F12-based reference medium was used as the cell culture medium, and PDL was calculated from the results of viable cell counts. PDL was 1.69 on day 7 of culture.
ＤＭＥＭ／Ｆ１２をベースとする基準培地１．５ｍＬを細胞培養液として使用する以外は実施例４に記載の手順と同様に、浮遊培養を行い、生細胞数の計測結果から、ＰＤＬを算出した。ＰＤＬは、培養７日目において１．６９であった。 [Comparative Example 3]
Suspension culture was performed in the same manner as in Example 4, except that 1.5 mL of a DMEM/F12-based reference medium was used as the cell culture medium, and PDL was calculated from the results of viable cell counts. PDL was 1.69 on day 7 of culture.
実施例４及び５と比較例３のＰＤＬの比較から、ジメチルオキサリルグリシン及び４－フェニル酪酸は、ｉＰＳ細胞の増殖性向上に有効であるとの結論を得た。
From the comparison of PDL in Examples 4 and 5 and Comparative Example 3, it was concluded that dimethyloxalylglycine and 4-phenylbutyric acid are effective in improving the proliferation of iPS cells.
本願の開示は、２０２１年３月１０日に出願された特願２０２１－３８７５３号及び２０２１年９月３０日に出願された特願２０２１－１６１５９６号に記載の主題と関連しており、それらの開示内容は引用によりここに援用される。
The disclosure of the present application relates to the subject matter described in Japanese Patent Application No. 2021-38753 filed on March 10, 2021 and Japanese Patent Application No. 2021-161596 filed on September 30, 2021, and those The disclosure is incorporated herein by reference.
Claims (13)
- アミノアルキルスルホン酸、アミノアルキルスルホン酸塩、カルボン酸鉄塩、ジメチルオキサリルグリシン、４－フェニル酪酸、及び、４－フェニル酪酸塩からなる群から選択される少なくとも１種を含有する、培地組成物。 A medium composition containing at least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, iron carboxylate, dimethyloxalylglycine, 4-phenylbutyric acid, and 4-phenylbutyrate.
- 前記アミノアルキルスルホン酸を含有する、請求項１に記載の培地組成物。 The medium composition according to claim 1, containing the aminoalkylsulfonic acid.
- 前記アミノアルキルスルホン酸が、２－アミノエチルスルホン酸を含む、請求項２に記載の培地組成物。 The medium composition according to claim 2, wherein said aminoalkylsulfonic acid comprises 2-aminoethylsulfonic acid.
- 前記カルボン酸鉄塩を含有する、請求項１～３のいずれか１項に記載の培地組成物。 The medium composition according to any one of claims 1 to 3, which contains the iron carboxylate.
- 前記カルボン酸鉄塩が、クエン酸鉄アンモニウムを含む、請求項４に記載の培地組成物。 The medium composition according to claim 4, wherein the iron carboxylate salt comprises ferric ammonium citrate.
- 前記アミノアルキルスルホン酸と前記カルボン酸鉄塩とを含有する、請求項１に記載の培地組成物。 The medium composition according to claim 1, comprising the aminoalkylsulfonic acid and the iron carboxylate.
- 前記アミノアルキルスルホン酸及び前記カルボン酸鉄塩がそれぞれ、２－アミノエチルスルホン酸及びクエン酸鉄アンモニウムを含む、請求項６に記載の培地組成物。 The medium composition of claim 6, wherein said aminoalkylsulfonic acid and said carboxylic acid iron salt comprise 2-aminoethylsulfonic acid and ferric ammonium citrate, respectively.
- 前記ジメチルオキサリルグリシンを含有する、請求項１～７のいずれか１項に記載の培地組成物。 The medium composition according to any one of claims 1 to 7, which contains the dimethyloxalylglycine.
- 前記４－フェニル酪酸又は前記４－フェニル酪酸塩を含有する、請求項１～８のいずれか１項に記載の培地組成物。 The medium composition according to any one of claims 1 to 8, which contains the 4-phenylbutyric acid or the 4-phenylbutyric acid salt.
- 細胞の浮遊培養に用いられる、請求項１～９のいずれか１項に記載の培地組成物。 The medium composition according to any one of claims 1 to 9, which is used for cell suspension culture.
- 多能性幹細胞の培養に用いられる、請求項１～１０のいずれか１項に記載の培地組成物。 The medium composition according to any one of claims 1 to 10, which is used for culturing pluripotent stem cells.
- 請求項１～１１のいずれか１項に記載の培地組成物を用いて、細胞を培養することを含む、細胞培養物の製造方法。 A method for producing a cell culture, comprising culturing cells using the medium composition according to any one of claims 1 to 11.
- 請求項１～１１のいずれか１項に記載の培地組成物と、多能性幹細胞とを含有する、細胞懸濁物。 A cell suspension containing the medium composition according to any one of claims 1 to 11 and pluripotent stem cells.
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| JP2021038753 | 2021-03-10 | ||
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| JP2021-161596 | 2021-09-30 |
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