WO2025018336A1 - 無血清培地 - Google Patents

無血清培地 Download PDF

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WO2025018336A1
WO2025018336A1 PCT/JP2024/025483 JP2024025483W WO2025018336A1 WO 2025018336 A1 WO2025018336 A1 WO 2025018336A1 JP 2024025483 W JP2024025483 W JP 2024025483W WO 2025018336 A1 WO2025018336 A1 WO 2025018336A1
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medium
serum
cholesterol
cells
2ilif
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French (fr)
Japanese (ja)
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大治 岡村
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Kindai University
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Kindai University
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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; 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

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  • the present invention relates to a medium for culturing ES cells, which is a serum-free medium that does not use fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • ES cells Mouse embryonic stem cells (ES cells), derived from the undifferentiated cell population of a preimplantation embryo (blastocyst), have pluripotency, self-renewal and unlimited proliferation capabilities, and are widely used as a model system for basic research on pluripotent stem cells and the development of cell therapy.
  • Fetal bovine serum contains over 1,000 components necessary for maintaining stable proliferation and viability of many cultured cells, including not only mouse ES cells but also cancer cell lines. Therefore, even today, many researchers still culture mouse ES cells in FBS-containing medium.
  • KSR medium containing Knockout Serum Replacement
  • N2B27 medium with the addition of N2 and B27 supplements
  • KSR and B27 supplements are expensive and their ingredients are not disclosed, so even though they are chemically defined media, there remains the issue that the medium composition cannot be controlled according to the researcher's purpose.
  • Patent Document 1 discloses a method for producing target cells, which includes culturing source cells in a serum-free medium containing at least one culture factor selected from a growth factor and a differentiation-inducing factor, human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin, to produce the target cells.
  • a serum-free medium containing at least one culture factor selected from a growth factor and a differentiation-inducing factor, human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin, to produce the target cells.
  • Patent Document 1 does not use FBS, and the components are also known. However, there is a high demand for a medium that is less expensive and has a high culture effect.
  • the present invention was conceived in consideration of the above problems, and provides a serum-free medium with fewer components and at a lower cost.
  • the serum-free medium comprises: Dulbecco's modified Eagle's medium (DMEM); Albumin (BSA), Insulin-transferrin-selenium-ethanolamine (ITS-X); It is characterized by containing cholesterol.
  • DMEM Dulbecco's modified Eagle's medium
  • BSA Albumin
  • ITS-X Insulin-transferrin-selenium-ethanolamine
  • the serum-free medium of the present invention makes it possible to culture ES cells using only a few ingredients: DMEM, which is a common medium, albumin (BSA), and insulin-transferrin-selenium-ethanolamine (ITS-X).
  • DMEM which is a common medium
  • BSA albumin
  • ITS-X insulin-transferrin-selenium-ethanolamine
  • the serum-free medium of the present invention can provide an inexpensive and stable cell culture environment.
  • DARP medium is composed of DA-X medium + ⁇ -ME + cholesterol (10 ⁇ M).
  • the serum-free medium of the present invention contains Dulbecco's modified Eagle's medium (DMEM), which is a basal medium, albumin (BSA), and insulin-transferrin-selenium-ethanolamine (ITS-X).
  • DMEM Dulbecco's modified Eagle's medium
  • BSA albumin
  • ITS-X insulin-transferrin-selenium-ethanolamine
  • DMEM only needs to have increased amounts of amino acids and vitamins compared to the original EME (Eagle's medium), and the contents of other components may differ by about 10%. Furthermore, the serum-free medium of the present invention does not need to contain F-12 Ham. However, this does not exclude the inclusion of F-12 Ham.
  • Albumin can also be used from a wide variety of sources. More specifically, it can be egg albumin, serum albumin, milk albumin, etc. Human serum albumin is more preferable.
  • Insulin-transferrin-selenium-ethanolamine may be any commercially available product, and a suitable composition for example would be insulin 1000.0 mg/L, transferrin 550.0 mg/L, selenium 173.0 mg/L, and ethanolamine 61.0 mg/L.
  • the albumin content in DMEM should be 3 mg/mL to 8 mg/mL, preferably 4 mg/mL to 7 mg/mL, and most preferably 4.5 mg/mL to 5.5 mg/mL.
  • ITS-X is preferably at 4.0 mg/mL to 7.0 mg/mL relative to DMEM, more preferably at 4.5 mg/mL to 6.5 mg/mL, and most preferably at 5.0 mg/mL to 6.0 mg/mL.
  • the above serum-free medium according to the present invention is called "DA-X medium.”
  • Cholesterol can be added to DA-X medium. Cholesterol only needs to be purified, and may be derived from a living organism or may be synthetic. Cholesterol should be 5 ⁇ M to 20 ⁇ M in DMEM, preferably 10 ⁇ M to 20 ⁇ M, and most preferably 10 ⁇ M.
  • DA-X medium containing cholesterol is called DARP medium.
  • the serum-free medium of the present invention can be used primarily for culturing ES cells, but we will also mention extracellular matrix proteins that serve as scaffolds during cell culture. Cells need a scaffold to grow. There are several proteins that can serve as scaffolds, and we have found that the serum-free medium of the present invention uses laminin as a scaffold to allow ES cells to grow optimally.
  • Mouse embryonic fibroblast (MEF) feeder cells were prepared from E14.5 mouse embryos.
  • E14tg2a mouse embryonic stem cell (ESC) line derived from 129/Ola (RIKEN BRC, AES0135) was maintained on Mitomycin C (MMC) (Fujifilm, 139-18711) inactivated MEF feeder cells in a single well of a 4-well plate (SPL Life Sciences, SPL-30004) in DF10LIF medium.
  • MMC Mitomycin C
  • the DF10LIF medium had the following composition: The medium was prepared by adding 10% fetal bovine serum (Gibco, 10270-106)-containing DMEM medium (Nacalai Tesque, 08458-45) containing recombinant mouse LIF (1x10 3 units/ml, Nacalai Tesque, NU0012-2), 1x MEM non-essential amino acid solution (NEAA) (Nacalai Tesque, 06344-58), 100 ⁇ M ⁇ -mercaptoethanol ( ⁇ -ME, Nacalai Tesque, 21438-82), 2mM L-glutamine (Nacalai Tesque, 16948-04), and 1x Penicillin-Streptomycin Mixed. Solution (P/S) (Nacalai Tesque, 26253-84) was prepared by mixing the above ingredients.
  • the medium was changed every 2 days, and colony-forming ESCs were dissociated with TrypLE (Gibco, 12604-013), subcultured on freshly prepared MMC-inactivated MEF feeder cells, and further cultured at a split ratio of 1:20 every 4 to 5 days.
  • TrypLE Gibco, 12604-013
  • DK10 medium DMEM (Nacalai Tesque, 08458-45) containing 10% knockout serum replacement (KSR, Gibco, 10828028) supplemented with 1x MEM non-essential amino acid solution (NEAA), 100 ⁇ M ⁇ -mercaptoethanol, 2 mM L-glutamine, and 1x penicillin-streptomycin mixed solution (P/S).
  • DMEM Non-essential amino acid solution
  • P/S penicillin-streptomycin mixed solution
  • N2B27 medium Neurobasal medium (Gibco, 21103049) and DMEM Ham's F-12 (Nacalai Tesque, 11581-15) mixed at a 1:1 ratio, with addition of N-2 supplement (1x, Gibco, 17502048), B-27 supplement (1x, Gibco, 17504044), and 2mM L-glutamine solution.
  • extracellular matrix Prior to seeding ESCs, extracellular matrix was precoated onto a single well of a 4-well cell culture plate at the desired concentration as described below, incubated at 37°C for 1 hour, and then aspirated for use: 2.35 ⁇ g/cm 2 fibronectin (Sigma-Aldrich, F1141), 1.25 ⁇ g/cm 2 vitronectin (FUJIFILM, 220-02041), 0.5 ⁇ g/cm 2 recombinant laminin 511-E8 fragment (iMatrix-511, MAX, 892011), 2% Matrigel matrix (Corning, 354234), 0.2% gelatin (Sigma, G1890), and 0.2% collagen type I (FUJIFILM, ASC-1-100).
  • DA-X medium consists of DMEM medium (Nacalai Tesque, 08458-45) supplemented with 1x ITS-X (Insulin-Transferrin-Selenium-Ethanolamine, Gibco, 51500056), 5 mg/mL BSA (Bovine Serum Albumin, Sigma, A3059), 1x L-glutamine, and 1x Penicillin-Streptomycin Mixed Solution.
  • DARP DA-X medium-based for Rodent Pluripotent stem cells
  • DA-X medium is DA-X medium to which 100 ⁇ M ( ⁇ -mercaptoethanol, ⁇ -ME) and 10 ⁇ M water-soluble cholesterol solution (a complex of methyl- ⁇ -cyclodextrin and cholesterol/EtOH solution, described below) have been added.
  • water-soluble cholesterol was administered in the range of 5-25 ⁇ M.
  • 3 ⁇ M CHIR99021 and 1 ⁇ M PD0325901 were supplemented as 2i and in combination with recombinant mouse LIF (1 ⁇ 10 3 units/ml) as 2iLIF. All medium components, including 2iLIF except cholesterol, were premixed, but only water-soluble cholesterol solution was added to the medium immediately before seeding the cells to prevent crystallization during storage at 4°C.
  • DA-X medium supports the proliferation of mouse ESCs using 2iLIF and ⁇ -mercaptoethanol
  • DA-X medium can support not only adherent cancer cell lines but also mouse pluripotent stem cell lines
  • To minimize the medium composition we first tested DA-X medium containing 2i and 2iLIF without other components, as these have been reported to be sufficient components to maintain mouse ESCs in an undifferentiated state.
  • DA-X 2iLIF medium Alkaline phosphatase staining showed a weak signal in DA-X 2i medium, indicating that the addition of LIF is essential for maintaining the undifferentiated state and proliferation of ESCs under DA-X medium conditions.
  • DA-X 2iLIF medium could not maintain cell viability and proliferation even after one passage. Therefore, we tested ⁇ -mercaptoethanol ( ⁇ -ME), an antioxidant widely used in the culture of pluripotent stem cells, under DA-X 2iLIF medium conditions, and found that it showed clear dome-shaped colony morphology and was effective in the proliferation of ESCs beyond passage.
  • ⁇ -ME ⁇ -mercaptoethanol
  • Extracellular matrix is a key component in regulating cell proliferation and differentiation not only in vivo but also in vitro cell culture systems. We aimed to identify specific extracellular matrices that could support proliferation and cell viability of undifferentiated ESCs over long-term culture passages under DA-X 2iLIF/ ⁇ -ME conditions.
  • FIG. 1 Several extracellular matrices that are widely used for efficient maintenance of human ESCs/iPS and mouse ESCs in an undifferentiated state were tested. The results are shown in Figure 1.
  • the horizontal axis indicates the number of days in culture, and the vertical axis indicates the number of cells.
  • the circle ( ⁇ ) in the graph indicates laminin-coated plates, the diamond ( ⁇ ) indicates gelatin-coated plates, the cross ( ⁇ ) indicates fibronectin-coated plates, the square ( ⁇ ) indicates materigel-coated plates, the triangle ( ⁇ ) indicates vitronectin-coated plates, and the cross (+) indicates the results for collagen-coated plates.
  • the horizontal axis represents the number of days of culture, and the vertical axis represents the number of cells.
  • triangles ( ⁇ ) represent the results when cholesterol was 25 ⁇ M
  • crosses ( ⁇ ) represent the results when cholesterol was 20 ⁇ M
  • circles ( ⁇ ) represent the results when cholesterol was 10 ⁇ M
  • squares ( ⁇ ) represent the results when cholesterol was 5 ⁇ M
  • diamonds ( ⁇ ) represent the results when there was no cholesterol.
  • DA-X 2iLIF/ ⁇ -ME medium containing 10 ⁇ M cholesterol is referred to as DARP 2iLIF medium (DA-X-based medium for Rodent Pluripotent stem cells).
  • DARP 2iLIF medium To maximize the proliferation potential in DARP 2iLIF medium, we focused on cholesterol and NEAAs in ESC proliferation in DARP 2iLIF medium. NEAAs also play important roles in various cellular processes, including proliferation of mouse ESCs. To investigate the effects of cholesterol and NEAAs on ESC proliferation in DARP 2iLIF medium, we performed proliferation curves over 30 days of continuous passages in comparison with other media defined for mouse ESCs, DK10 2iLIF medium and N2B27 2iLIF medium. The results are shown in Figure 3.
  • the horizontal axis indicates the number of days of culture, and the vertical axis indicates the number of cells.
  • black squares ( ⁇ ) indicate the results in the case of DK10 2iLIF
  • black circles ( ⁇ ) indicate the results in the case of DARP 2iLIF
  • black triangles ( ⁇ ) indicate the results in the case of DARP 2iLIF _NEAA(+)
  • black diamonds ( ⁇ ) indicate the results in the case of DARP 2iLIF _NEAA(+)_Cholesterol(-)
  • crosses ( ⁇ ) indicate the results in the case of DARP 2iLIF _Cholesterol(-)
  • squares ( ⁇ ) indicate the results in the case of N2B27 2iLIF on Laminin
  • circles ( ⁇ ) indicate the results in the case of N2B27 2iLIF on Gelatin.
  • DARP 2iLIF medium A characteristic of ESCs maintained in DARP 2iLIF medium was that the colony morphology of ESCs was observed as a unique amoeba-like monolayer rather than the conventional dome-like one.
  • cholesterol-containing DARP 2iLIF medium achieved robust ESC proliferation comparable to or better than other chemically defined media due to its unique colony morphology.
  • the serum-free medium of the present invention can be suitably used for culturing mouse ES cells, which are naive pluripotent stem cells.

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PCT/JP2024/025483 2023-07-20 2024-07-16 無血清培地 Pending WO2025018336A1 (ja)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09505462A (ja) * 1993-08-23 1997-06-03 バクスター、インターナショナル、インコーポレイテッド 無血清培地内での好中球前駆細胞及び巨核球前駆細胞の試験管内増殖
JP2002529071A (ja) * 1998-11-09 2002-09-10 コンソルツィオ・ペール・ラ・ジェスティオーネ・デル・チェントロ・ディ・ビオテクノロジー・アヴァンツァテ 軟骨細胞様細胞のための無血清培地
JP2005515777A (ja) * 2002-01-25 2005-06-02 ジェンザイム・コーポレーション 軟骨細胞のための無血清培地およびその使用法
JP2009535058A (ja) * 2006-05-02 2009-10-01 ウイスコンシン アラムニ リサーチ ファンデーション 幹細胞の内胚葉細胞および膵臓系列細胞への分化方法
JP2014519837A (ja) * 2011-06-23 2014-08-21 タムペレーン イリオピスト インビトロの心臓血管モデル
JP2020508687A (ja) * 2017-03-09 2020-03-26 エボニック オペレーションズ ゲーエムベーハー ジペプチドを含む培地
WO2021085398A1 (ja) * 2019-10-28 2021-05-06 第一三共株式会社 Ｃｄ４５ｒａ＋およびｃｃｒ７＋の細胞表面マーカーを有するｔ細胞の製造方法
WO2023021591A1 (ja) * 2021-08-18 2023-02-23 国立大学法人東北大学 子宮内膜モデル、子宮内膜モデルの作製方法、及び子宮内膜着床モデル

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09505462A (ja) * 1993-08-23 1997-06-03 バクスター、インターナショナル、インコーポレイテッド 無血清培地内での好中球前駆細胞及び巨核球前駆細胞の試験管内増殖
JP2002529071A (ja) * 1998-11-09 2002-09-10 コンソルツィオ・ペール・ラ・ジェスティオーネ・デル・チェントロ・ディ・ビオテクノロジー・アヴァンツァテ 軟骨細胞様細胞のための無血清培地
JP2005515777A (ja) * 2002-01-25 2005-06-02 ジェンザイム・コーポレーション 軟骨細胞のための無血清培地およびその使用法
JP2009535058A (ja) * 2006-05-02 2009-10-01 ウイスコンシン アラムニ リサーチ ファンデーション 幹細胞の内胚葉細胞および膵臓系列細胞への分化方法
JP2014519837A (ja) * 2011-06-23 2014-08-21 タムペレーン イリオピスト インビトロの心臓血管モデル
JP2020508687A (ja) * 2017-03-09 2020-03-26 エボニック オペレーションズ ゲーエムベーハー ジペプチドを含む培地
WO2021085398A1 (ja) * 2019-10-28 2021-05-06 第一三共株式会社 Ｃｄ４５ｒａ＋およびｃｃｒ７＋の細胞表面マーカーを有するｔ細胞の製造方法
WO2023021591A1 (ja) * 2021-08-18 2023-02-23 国立大学法人東北大学 子宮内膜モデル、子宮内膜モデルの作製方法、及び子宮内膜着床モデル

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* Cited by examiner, † Cited by third party
Title
KOLKMANN ANNA M., VAN ESSEN ANON, POST MARK J., MOUTSATSOU PANAGIOTA: "Development of a Chemically Defined Medium for in vitro Expansion of Primary Bovine Satellite Cells", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, vol. 10, XP093076763, DOI: 10.3389/fbioe.2022.895289 *
TAKII, SHINO, OKAMURA, DAIJI: "P-11-8-6 Development of a Universal Serum-Free Culture Medium in Cancer Cell Lines – The Importance of Cholesterol Metabolism", THE 80TH ANNUAL MEETING OF THE JAPANESE CANCER ASSOCIATION; SEPTEMBER 30 - OCTOBER 2, 2021, JAPANESE CANCER ASSOCIATION, JP, vol. 80, 2 October 2021 (2021-10-02) - 2 October 2021 (2021-10-02), JP, XP009561371 *

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