Culture medium composition, method for producing cell cultures, and cell suspension

Abstract

The present invention provides a culture medium composition that can improve the efficiency of cell proliferation. [Solution] This disclosure relates to a culture medium composition comprising at least one selected from the group consisting of aminoalkyl sulfonic acid, aminoalkyl sulfonate, and iron ammonium carboxylate.

Description

[[Technical Field]]

[0001] The present disclosure relates to a medium composition, a method for producing a cell culture, and a cell suspension. [[Background Art]]

[0002] Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have proliferation ability and differentiation ability, and thus are promising as raw materials for preparations administered in cell therapy, regenerative medicine, and the like. For the practical application of cell therapy and regenerative medicine, it is required that a necessary amount of pluripotent stem cells, which are raw materials, be stably supplied.

[0003] For industrially controlling cell culture, the composition of the medium composition greatly affects cell productivity. Components contained in the medium composition are selected according to the cell type or the purpose of culture.

[0004] For example, Non-Patent Document 1 reports that when iPS cells and cardiomyocytes differentiation-induced from iPS cells are co-cultured in a culture solution containing a high concentration of lactic acid, cardiomyocytes selectively proliferate, and thus purification of iPS cell-derived cardiomyocytes can be achieved.

[0005] Non-Patent Document 2 reports that in undifferentiated maintenance culture of iPS cells, increasing the tryptophan concentration in a culture solution improves the proliferation of iPS cells.

[0006] Patent Document 1 discloses that addition of ethanolamine is effective for promoting proliferation of pluripotent stem cells. [[Prior Art Literature]] [[Patent Literature]]

[0007] [[Patent Document 1]] International Publication No. 2014 / 119219 [[Non-Patent Literature]]

[0008] [Non-Patent Document 1] “Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes,”Cell Stem Cell (2013) [Non-Patent Document 2] “Tryptophan Metabolism Regulates Proliferative Capacity of Human Pluripotent Stem Cells,” iSCIENCE (2021) [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] For cell cultures used as raw materials for cell therapy and regenerative medicine, it is recommended from a safety standpoint not to use heterogeneous raw materials in the culture process, and it is desirable that the culture medium be a completely synthetic medium.

[0010] However, compared to culture media using heterogeneous raw materials, there is room for improvement in terms of cell proliferation efficiency when culturing in completely synthetic media. In particular, for pluripotent stem cells such as ES cells and iPS cells, there is a challenge in optimizing the composition of the culture medium to improve culture efficiency.

[0011] This disclosure provides a culture medium composition that can improve the cell proliferation efficiency. This disclosure provides a method for producing a cell culture that can efficiently grow cells. This disclosure provides a cell suspension that can efficiently grow cells. [Means for solving the problem]

[0012] Examples of embodiments are given below. The present invention is not limited to the following embodiments.

[0013] (1) A culture medium composition containing at least one selected from the group consisting of aminoalkyl sulfonic acid, aminoalkyl sulfonate, and ironammonium carboxylate. (2) The culture medium composition according to (1) above, wherein the content of the aminoalkyl sulfonic acid is 50 to 200 μg / mL based on the total volume of the culture medium composition. (3) The culture medium composition according to (1) or (2) above, wherein the content of iron ammonium carboxylate is 0.1 to 1 μg / mL based on the total volume of the culture medium composition. (4) The culture medium composition according to any one of (1) to (3) above, wherein the culture medium composition contains the aminoalkyl sulfonic acid, and the aminoalkyl sulfonic acid contains 2-aminoethylsulfonic acid. (5) The culture medium composition according to any one of (1) to (4) above, wherein the culture medium composition contains iron ammonium carboxylate, and the iron ammonium carboxylate contains iron ammonium citrate. (6) A culture medium composition according to any one of the above items (1) to (5), used for suspension culture of cells. (7) A culture medium composition according to any one of the above items (1) to (6), used for culturing pluripotent stem cells. (8) A method for producing a cell culture, comprising culturing cells using the culture medium composition described in any one of the above items (1) to (7). (9) A method for producing a cell culture according to (8) above, wherein the cells are cultured in suspension. (10) A method for producing a cell culture according to (8) or (9) above, wherein the cells include pluripotent stem cells. (11) A cell suspension containing the culture medium composition described in any one of items (1) to (7) above, and pluripotent stem cells. [Effects of the Invention]

[0014] According to the present disclosure, a medium composition capable of improving cell proliferation efficiency is obtained. According to the present disclosure, a method for producing a cell culture that enables efficient cell proliferation is obtained. According to the present disclosure, a cell suspension that enables efficient cell proliferation is obtained. Mode for Carrying Out the Invention

[0015] Embodiments of the present invention will be described. The present invention is not limited to the following embodiments. In the present specification, a numerical range indicated by using "~" indicates a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In the numerical ranges described stepwise in the present specification, the upper limit or lower limit of the numerical range at one stage can be arbitrarily combined with the upper limit or lower limit of the numerical range at another stage. Unless otherwise specified, the materials exemplified in the present specification can be used alone or in combination of two or more. In the present specification, when a plurality of substances corresponding to each component are present in the composition, the content of each component in the composition means the total amount of the plurality of substances present in the composition, unless otherwise specified. The term "step" includes not only an independent step, but also a case where the desired effect of the step is achieved even if it cannot be clearly distinguished from other steps.

[0016] <Medium Composition> The medium composition contains at least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, and iron ammonium carboxylate. At least one selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, and iron ammonium carboxylate is effective for improving the proliferation efficiency of a culture target. The medium composition may contain known components included in general medium compositions, in addition to aminoalkylsulfonic acid, aminoalkylsulfonate, and iron ammonium carboxylate.

[0017] 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. The liquid medium can contain water as a base material. The powder medium can be combined with water or the like at the time of use and used as a liquid medium or a gel medium. The powder medium is obtained by a known method such as drying after preparing a medium composition containing an aqueous medium.

[0018] The aminoalkylsulfonic acid may be a compound represented by NR'2-R-SO3H (R represents an alkylene group, and each R' independently represents a hydrogen atom or an alkyl group). The aminoalkylsulfonate is NR'2-R-SO3 - X + a compound represented by (R represents an alkylene group, each R' independently represents a hydrogen atom or an alkyl group, and X + represents a cation.).

[0019] The alkylene group may 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 to 8, or may be 1 to 4. The alkylene group includes, for example, at least one selected from the group consisting of methylene, ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, n-heptylene, n-octylene, isopropylene, isobutylene, sec-butylene, tert-butylene, 2-ethylhexylene, 3,7-dimethyloctylene, cyclohexylene, cycloheptylene, and cyclooctylene. The alkyl group may be a linear alkyl group, a branched alkyl group, or a cyclic alkyl group. The number of carbon atoms in the alkyl group may be, for example, 1 to 8, 1 to 4, or 1 or 2. The alkyl group includes, for example, at least one selected from the group consisting of 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-butyl group, tert-butyl group, 2-ethylhexyl group, cyclohexyl group, cycloheptyl group, and cyclooctyl group. + It includes at least one selected from the group consisting of sodium ions, potassium ions, and ammonium ions.

[0020] The aminoalkyl sulfonic acid includes, for example, at least one selected from the group consisting of aminomethylsulfonic acid, 2-aminoethylsulfonic acid, N-methyl-2-aminoethylsulfonic acid, N-ethyl-2-aminoethylsulfonic acid, 3-aminopropylsulfonic acid, 4-aminobutylsulfonic acid, 6-aminohexylsulfonic acid, and 8-aminooctylsulfonic acid.

[0021] An aminoalkyl sulfonate includes, for example, at least one selected from the group consisting of sodium aminoalkylsulfonate, potassium aminoalkylsulfonate, and ammonium aminoalkylsulfonate. An aminoalkyl sulfonate includes, for example, at least one selected from the group consisting of aminomethylsulfonate ion, 2-aminoethylsulfonate ion, N-methyl-2-aminoethylsulfonate ion, N-ethyl-2-aminoethylsulfonate ion, 3-aminopropylsulfonate ion, 4-aminobutylsulfonate ion, 6-aminohexylsulfonate ion, and 8-aminooctylsulfonate ion, and at least one selected from the group consisting of sodium ion, potassium ion, and ammonium ion.

[0022] The aminoalkyl sulfonic acid content in the culture medium composition may be, for example, 50-200 μg / mL, 100-150 μg / mL, or 110-140 μg / mL relative to the total volume of the culture medium composition, when it is a liquid medium or a gel medium. If the culture medium composition is a powder medium, the liquid medium or gel medium prepared from the powder medium should satisfy the above-mentioned range of aminoalkyl sulfonic acid content.

[0023] The content of aminoalkyl sulfonates should satisfy the range of aminoalkyl sulfonic acid content mentioned above when the cations are replaced with hydrogen ions.

[0024] Ammonium iron carboxylate has the anion -COO - The salt may contain a group and iron ions and ammonium ions as cations, and the anion R-(COO - )n may be an organic iron ammonium carboxylate containing iron ions and ammonium ions as cations (R represents a monovalent or greater organic group, and n represents an integer of 1 or greater. n is the -COO bonded to R -(This is the number of groups.) R is an atomic group obtained by removing one or more hydrogen atoms from a substituted or unsubstituted saturated hydrocarbon, and the saturated hydrocarbon may be a linear saturated hydrocarbon, a branched saturated hydrocarbon, or a cyclic saturated hydrocarbon. The number of carbon atoms in the saturated hydrocarbon may be, for example, 1 to 12, 1 to 8, or 1 to 6. The saturated hydrocarbon includes, for example, at least one selected from the group consisting of methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane. Examples of substituents that the saturated hydrocarbon may have include a hydroxyl group, an amino group, a monoalkylamino group (the number of carbon atoms in the alkyl group is, for example, 1 to 4), a dialkylamino group (the number of carbon atoms in the alkyl group is independently, for example, 1 to 4), etc. n may be, for example, 2 to 6, or 2 to 5.

[0025] The ammonium iron carboxylate includes, for example, at least one selected from the group consisting of ammonium iron citrate, ammonium iron butyrate, ammonium iron pyruvate, ammonium iron oxalate, ammonium iron fumarate, ammonium iron malate, ammonium iron succinate, ammonium iron diethylenetriaminepentaacetate, ammonium iron ethylenediaminetetraacetate, and ammonium iron dicarboxymethylglutamate. The culture medium composition may contain sodium iron carboxylate in place of, or together with, ammonium iron carboxylate.

[0026] The content of ammonium iron carboxylate in the culture medium composition may be, for example, 0.1 to 1 μg / mL, 0.2 to 0.5 μg / mL, or 0.2 to 0.4 μg / mL relative to the total volume of the culture medium composition, when it is a liquid medium or gel medium. If the culture medium composition is a powder medium, it is sufficient that the liquid medium or gel medium prepared from the powder medium satisfies the above range of ammonium iron carboxylate content.

[0027] The amount of sodium iron carboxylate should satisfy the above-mentioned range of ammonium iron carboxylate content when sodium ions are replaced with ammonium ions.

[0028] Optional additives that the culture medium composition may contain include inorganic salts, sugars, amino acids, proteins, serum, serum substitutes, vitamins, hormones, antibiotics, and growth factors. The culture medium composition may contain heterogeneous raw materials or may be a completely synthetic medium that does not contain heterogeneous raw materials. Even when the culture medium composition is a completely synthetic medium, it contains at least one selected from the group consisting of aminoalkyl sulfonic acid, aminoalkyl sulfonate, and iron ammonium carboxylate, which allows for efficient cell growth.

[0029] Culture medium compositions can be prepared, for example, by adding at least one substance selected from the group consisting of aminoalkylsulfonic acid, aminoalkylsulfonate, and ironammonium carboxylate to a known basal medium or other existing medium. Examples of known basal media include DMEM (Dulbeccoo's modified Eagle medium), MEM (Eagle's minimal essential medium), αMEM medium (Eagle's minimal essential medium α modified), GMEM (Glasgow's minimal essential medium), IMDM (Iskoff'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, FUJIFILM Irvine Scientific), Essential8 (Thermo Fisher Scientific), Essential6 (Thermo Fisher Scientific), and mixtures containing two or more substances selected from these.

[0030] Essential8 is a culture medium consisting of DMEM / F-12, L-ascorbic acid, selenium, transferrin, NaHCO3, insulin, FGF2, and TGFβ1, along with water. Essential6 is a culture medium consisting of DMEM / F-12, L-ascorbic acid, selenium, transferrin, NaHCO3, and insulin, along with water. Because Essential8 and Essential6 contain fewer components, they are easy to manage and reduce lot-to-lot variability in cultures.

[0031] The culture medium composition is not particularly limited and can be used for culturing various cells. The cells are preferably of animal origin, and more preferably of mammalian origin. Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, and cats. Examples of cells include pluripotent stem cells, somatic stem cells, and somatic cells. The cells may be pluripotent stem cells. Examples of pluripotent stem cells include ES cells, iPS cells, EG cells (Embryonic germ cells), and GS cells (Germ-line stem cells). Examples of somatic stem cells include mesenchymal stem cells and tissue stem cells.

[0032] The culture medium composition is not particularly limited and can be used as a culture medium for various culture methods or for the preparation of such culture media. Culture methods include two-dimensional culture, in which cells are cultured attached to a fixed plane, and three-dimensional culture, in which cells are cultured suspended in a culture medium. When performing three-dimensional culture, the cells may be in any form, suspended as single cells, cell aggregates, or cell masses, or suspended while attached to a culture carrier such as a microcarrier. In this disclosure, "single cell" means one independent cell.

[0033] <Methods for producing cell cultures, methods for culturing cells> A method for producing a cell culture includes culturing cells using the culture medium composition according to this disclosure. For culturing, a liquid medium or a gel medium can be used as the culture medium composition. The method of 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 culture medium composition. The cells to be cultured are 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 culture medium composition. This makes it possible to obtain a culture containing pluripotent stem cells and the culture medium composition according to this disclosure. That is, a culture according to this disclosure includes the culture medium composition according to this disclosure and cultured pluripotent stem cells.

[0034] The culture temperature may be 36.0-38°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% CO2 atmosphere.

[0035] The method for producing a cell culture may include any steps. Optional steps include, for example, thawing frozen cells, washing cells, seeding cells, adding a culture medium or at least one component contained in the culture medium to the cell culture, replacing at least a portion of the culture medium contained in the cell culture, allowing the cell culture to stand, separating and recovering the cells from the culture medium composition using a filter or the like after a predetermined culture period, and storing the recovered cells in combination with a preservation composition. Examples of preservation compositions include other known preservation media and cryopreservation compositions.

[0036] The cell culture method includes culturing cells using the culture medium composition. The above description of the method for producing cell cultures can also be applied to the cell culture method.

[0037] <Cell culture> A cell culture can be obtained by a method for producing a cell culture or a method for culturing cells. The cell culture comprises the culture medium composition and cells. After culturing, the proliferated cells can be recovered from the cell culture.

[0038] <Cell suspension> The cell suspension contains the culture medium composition according to this disclosure and pluripotent stem cells. The pluripotent stem cells may be pluripotent stem cells cultured using the culture medium composition according to this disclosure. By using this cell suspension as is for culturing pluripotent stem cells, it is possible to start or continue culturing pluripotent stem cells with higher culture efficiency than conventional methods.

[0039] Cells obtained by culturing using the culture medium composition relating to this disclosure may possess undifferentiated potential. The undifferentiated potential of cells can be confirmed by confirmation methods known in the industry. Methods for confirming undifferentiated potential include, for example, confirming the expression level or abundance of known factors such as genes, proteins, and glycolipids, either individually or in combination. Such factors may be referred to as markers, and genes and proteins that serve as markers may be referred to as marker genes and marker proteins, respectively.

[0040] Examples of markers include Nanog, Sox2, Oct-4, Klf4, Lin28, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, SSEA-1, SSEA-3, SSEA-4, and c-Myc. These marker factors can be used individually 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 measuring the expression level of marker genes by quantitative PCR, and confirming or measuring the presence or amount of surface antigens or other markers by immunoassays such as flow cytometry. Cells with undifferentiated potential may, depending on the cell type, origin, etc., tend to show increased levels of one or more 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, and c-Myc, compared to known cells that do not exhibit undifferentiated potential. Therefore, cells with undifferentiated potential can be identified, depending on the cell type, origin, etc., as cells that exhibit one or more characteristics such as high Nanog levels, high Sox2 levels, high Oct-4 levels, high Klf4 levels, high Lin28 levels, high TRA-1-60 levels, high TRA-1-81 levels, high TRA-2-49 / 6E levels, high SSEA-1 levels, high SSEA-3 levels, high SSEA-4 levels, and high c-Myc levels, compared to known cells that do not exhibit undifferentiated potential. For example, cells that exhibit one or more characteristics such as high Nanog levels, high Sox2 levels, high Oct-4 levels, and high SSEA-4 levels. [Examples]

[0041] Embodiments of the present invention will be specifically described by reference to examples. Embodiments of the present invention are not limited to the following examples.

[0042] [Example 1] As cells, we used human iPS cells (201B7 strain) provided by Kyoto University, a national university corporation.

[0043] (Preparation of cell culture medium (liquid medium)) Cell culture medium 1 was prepared by adding 50 μg of bFGF (R&D Systems) and 62.5 mg of 2-aminoethylsulfonic acid (taurine, Fujifilm Wako Pure Chemical Industries, Ltd.) to 500 mL of Essential6 (Thermo Fisher Scientific). The amount of bFGF added was 100 ng / mL, and the amount of 2-aminoethylsulfonic acid added was 125 μg / mL.

[0044] (Suspension culture of 201B7) Add 6 mL of cell culture medium 1 to a 6 cm petri dish (AGC Techno Glass Co., Ltd.) with a low-adhesion coating, then add 2.5 × 10¹³ 201B7. 5 Cells were seeded. Petri dishes were placed in an incubator at 37°C with a CO2 concentration of 5%, and suspension culture was performed. The culture was continued for 15 days, and the number of viable cells was measured on days 3, 7, 10, and 15 of the culture period.

[0045] (Measurement of the number of viable cells) The number of viable cells was measured according to the protocol using the NC-200 cell counting device (NucleoCounter®, ChemoMetec). A portion of the cell suspension after enzyme treatment was collected and aspirated using a Via1 cassette specifically designed for the cell counting device. The Via1 cassette was then placed in the NC-200 and the measurement was performed.

[0046] (Calculation of PDL) The population doubling level (PDL) was calculated from the number of viable cells obtained by measurement using the following formula. In the formula, UCY represents the cell yield, l represents the number of cells seeded for culture, and X represents the initial number of divisions of the seeded cell population. PDL = 3.32 (log UCY - log l) + X

[0047] [Example 2] Cell culture medium 2 was prepared by adding 1.5 μg of ammonium iron citrate (Fujifilm Wako Pure Chemical Industries, Ltd.) to 6 mL of cell culture medium 1 prepared as described in Example 1. The amount of bFGF added was 100 ng / mL, the amount of 2-aminoethylsulfonic acid added was 125 μg / mL, and the amount of ammonium iron citrate added was 0.25 μg / mL. Next, suspension culture was performed in the same manner as described in Example 1, and PDL was calculated from the measurement results of the number of viable cells.

[0048] [Comparative Example 1] A cell culture medium was prepared by adding 600 ng of bFGF (R&D Systems) to 6 mL of Essential6 (Thermo Fisher Scientific). Next, suspension culture was performed in the same manner as described in Example 1, and PDL was calculated from the measurement results of the number of viable cells.

[0049] (Comparison of PDLs) Table 1 shows the PDL calculation results for Examples 1 and 2 and Comparative Example 1.

[0050] [Table 1]

[0051] [Example 3] Cell culture medium 3 was prepared by adding 600 ng of bFGF (R&D Systems) and 1.5 μg of ammonium iron citrate (Fujifilm Wako Pure Chemical Industries, Ltd.) to 6 mL of Essential 6 (Thermo Fisher Scientific). Subsequently, suspension culture was performed in the same manner as described in Example 1, and the PDL was calculated from the measurement of the number of viable cells. The PDL was 3.38 on day 4 of culture.

[0052] [Comparative Example 2] Following the same procedure as described in Comparative Example 1, suspension culture was performed, and PDL was calculated from the measurement of viable cell counts. The PDL was 2.78 on day 4 of culture.

[0053] From a comparison of PDL levels in the examples and comparative examples, it was concluded that the addition of 2-aminoethylsulfonic acid and ferric ammonium citrate is effective in improving the proliferation of iPS cells.

Claims

1. A culture medium composition containing at least one selected from the group consisting of aminoalkyl sulfonic acid, aminoalkyl sulfonate, and iron ammonium carboxylate.

2. The culture medium composition according to claim 1, wherein the culture medium composition contains the aminoalkyl sulfonic acid, and the aminoalkyl sulfonic acid comprises 2-aminoethylsulfonic acid.

3. The culture medium composition according to claim 1 or 2, wherein the culture medium composition contains iron ammonium carboxylate, and the iron ammonium carboxylate contains iron ammonium citrate.

4. A culture medium composition according to any one of claims 1 to 3, used for suspension culture of cells.

5. A culture medium composition according to any one of claims 1 to 4, used for culturing pluripotent stem cells.

6. A method for producing a cell culture, comprising culturing cells using the culture medium composition described in any one of claims 1 to 5.

7. A cell suspension comprising the culture medium composition according to any one of claims 1 to 5 and pluripotent stem cells.

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