WO2003097819A1 - Methode de culture cellulaire au moyen d'eau de mer des grands fonds - Google Patents

Methode de culture cellulaire au moyen d'eau de mer des grands fonds Download PDF

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Publication number
WO2003097819A1
WO2003097819A1 PCT/JP2003/003941 JP0303941W WO03097819A1 WO 2003097819 A1 WO2003097819 A1 WO 2003097819A1 JP 0303941 W JP0303941 W JP 0303941W WO 03097819 A1 WO03097819 A1 WO 03097819A1
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WO
WIPO (PCT)
Prior art keywords
cell culture
cells
culture method
medium
cell
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Application number
PCT/JP2003/003941
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English (en)
Japanese (ja)
Inventor
Yasuyuki Ishizuka
Motoaki Imamura
Original Assignee
Applied Cell Biotechnologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Applied Cell Biotechnologies, Inc. filed Critical Applied Cell Biotechnologies, Inc.
Priority to AU2003236182A priority Critical patent/AU2003236182A1/en
Publication of WO2003097819A1 publication Critical patent/WO2003097819A1/fr

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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N5/0018—Culture media for cell or tissue culture
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts

Definitions

  • the present invention relates to cell culture technology.
  • the present invention relates to a cell culture method using deep ocean water and a technique related to the method, which are useful in the field of regenerative medicine and the like.
  • the animal cell culture method established at the beginning of the 20th century is a technology in which the minimum amount of nutrients such as amino acids necessary for cells is added to the cells in the culture vessel and proliferated. This is a technique that utilizes the property that, based on the chemotaxis (chemotaxis) of the cells, the cells gradually migrate to the surrounding area while using nutrients to proliferate.
  • chemotaxis chemotaxis
  • cells are classified into “self-cultured cells” and “homogeneous cultured cells” depending on whether or not the cells are autologous.
  • the culture conditions are required because there are many types of growth factors required, and the growth factors required also vary depending on the origin of the cells to be cultured and the individual. It is diverse. For this reason, it has been difficult to set uniform and accurate culture conditions. That is, the conventional method of adding the selected minimum nutrients (vitamin, protein, etc.) to the culture solution forms a medium that satisfies all of the various culture conditions required by various cells. could not. For this reason, when culturing cells such as normal cells, by adding vitamins and various proteins to the basic medium, it is necessary to adjust the culturing conditions under which the cells intended for culturing can be proliferated and to prepare them. I had to rub the law. .
  • the present invention provides a novel cell culture medium that satisfies various culture conditions required by cells, and provides a cell culture method using the cell culture solution and an autoculture method based on the cell culture method.
  • the main purpose is to Disclosure of the invention
  • the present application provides the following method relating to cell culture.
  • a cell culture method using deep ocean water as a cell culture solution is provided. Specifically, it is possible to provide a method of using deep sea water as a substitute for distilled water for producing a cell culture solution, and a method of using deep sea water as a substitute nutrient for added nutrients of a cell culture solution.
  • deep ocean water generally refers to seawater at a depth of more than 200 meters, and a vertically sinking earth called “plum” caused by a difference in salinity around Greenland. It is a seawater that is said to have been around the Earth for centuries without ever touching the atmosphere since the current of the scale began.
  • This deep ocean water is hardly exposed to sunlight, and is in a state of low temperature and high pressure, so it contains abundant inorganic nutrients (minerals) with few bacteria.
  • the cell culture method according to the present invention is characterized in that this “deep ocean water” is actively used as a cell culture medium (medium).
  • the cell culture method according to the present invention is a method that is very reasonable especially for culturing normal cells.
  • the cell culture method according to the present invention is applied to an autologous culture method for culturing normal human cells under serum-free conditions, the risk of infection by non-autologous viruses or prions can be fundamentally eliminated. Therefore, it is a safe cell culture technology useful in the fields of regenerative medicine and the like.
  • the present invention has a technical significance of providing a technology relating to a cell culture method having versatility to cells and an autoculture method based on the method to related industries.
  • FIG. 1 is a diagram (table) showing the measurement results of cell proliferation in Experiment 1.
  • Figure 2 is an electron micrograph (20% by weight of deep seawater medium) showing the results of Experiment 2.
  • Fig. 3 is an electron micrograph (30% by weight of deep seawater medium) showing the results of Experiment 2.
  • Fig. 4 is an electron micrograph (40% by weight of deep seawater medium) showing the results of Experiment 2.
  • FIG. 5 is an electron micrograph (50% by weight of deep seawater medium) showing the results of Experiment 2.
  • Fig. 6 is an electron micrograph (60% by weight of deep seawater medium) showing the results of Experiment 2. '
  • FIG. 7 is an electron micrograph (70% by weight of deep seawater medium) showing the results of Experiment 2.
  • FIG. 8 is a diagram (table) showing the measurement results of cell proliferation in Experiment 3.
  • a powdered DMEM (Dulbecco's Modified Eagle Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.) is dissolved in deep ocean water in advance, sterilized by autoclave, and antibiotics (final concentration, penicillin: 50 units / ml, streptomycin: 50 g) / ml), glutamic acid (final concentration, 3.6 mM), sodium bicarbonate (final concentration, 0.09%), and fetal bovine serum (final concentration: 10%) (hereinafter referred to as “FBS”).
  • DMEM medium containing deep sea water was prepared by adding it to the final concentration.
  • the deep sea water-containing DMEM medium was added to the deep sea water at a concentration of S, 1, 5, 10, 20, and 30% by weight, and the deep sea water-containing DMEM medium was not added.
  • the cells were cultured in a 37 ° C incubator together with the added sample. After culturing for 2 days, the proliferation of the cells was measured using MTT Atssay (WST-1: Takara).
  • the proliferation of the cells can be represented by the absorbance at 450 nm. The higher the cell proliferation, the higher the absorbance.
  • the concentration of deep sea water added to the medium is preferably in the range of :! to 30% by weight, and particularly preferably in the range of 10 to 20% by weight.
  • the above-mentioned medium was replaced with a medium containing 20, 30, 40, 50, 60, and 70% by weight of the deep sea water, and cultured.
  • the state of culture on the following day was observed and photographed with an electron microscope, and the photographs were shown in FIGS. 2 to 7.
  • cells were observed to be killed in a medium containing 40 to 70% by weight of deep sea water, but in a medium containing 20 to 30% by weight of deep sea water, cells survived well. It was found to be growing.
  • Powdered DMEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was dissolved in the same deep sea water as in Experiments 1 and 2, autoclaved, and antibiotics (final concentration, penicillin: 50 units / ml, streptomycin: 50 ⁇ gZml) , Glutamic acid (final concentration, 3.6 mM) and sodium hydrogencarbonate (final concentration, 0.09%) were added to the respective final concentrations to prepare a serum-free, deep-sea marine DMEM medium.
  • antibiotics final concentration, penicillin: 50 units / ml, streptomycin: 50 ⁇ gZml
  • Glutamic acid final concentration, 3.6 mM
  • sodium hydrogencarbonate final concentration, 0.09%
  • Human primary fibroblasts (human normal cells) 96 Ueru culture plates IX 10 3 cells / Ueru: were seeded in 5% FBS-containing DMEM medium (Nissui Pharmaceutical Co., Ltd.), the next day After washing three times with PBS (-), the medium was replaced with a serum-free 'Deepwater ZDMEM medium', and the culture was continued in a 37 ° C incubator. After culturing for 2 days, the proliferation of the cells was measured by MTT Atssay (WST-1: Takara). The results are shown in Figure 8 (table).
  • Fig. 8 As shown in Fig. 8 (Table), cell growth was promoted by 13%, 21%, and 18% in a serum-free medium containing 5, 10, and 20% by weight of deep sea water. However, in the serum-free medium containing 30% by weight of deep sea water, cell growth was conversely inhibited.
  • the cell culture method using the deep sea water as a substitute for distilled water for preparing the cell culture solution or as an alternative nutrient for the added nutrient in the cell culture solution, and the deep sea water It has been found that an autologous culture method for growing normal human cells can be provided in a serum-free medium containing.
  • cell growth can be promoted by performing cell culture using a cell culture solution (medium) containing deep ocean water.
  • deep-sea water contains a variety of mineral components in a well-balanced manner and is applicable to the cultivation of a wide variety of cells
  • the cell culture method according to the present invention can be applied to an autologous culture method for culturing normal human cells under serum-free conditions, and the autologous culture method can reduce the risk of infection by non-autologous virus sprion and the like. No cell culture can be performed. Therefore, it is a useful technology for regenerative medicine. Specifically, medical materials, cosmetics, and pharmaceuticals that utilize collagen or other cell-producing components produced in cells or cultures cultured under the autologous culture method described above are safe from infection and safe. Furthermore, it can be used for research and development of medical materials, cosmetics, pharmaceuticals, etc. dedicated to self without concern about allergic reactions and rejection reactions.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Cette invention a trait à une méthode de culture cellulaire qui satisfait diverses conditions de cultures requises par des cellules et qui est donc largement utilisable. Notamment, ladite invention concerne une méthode de culture cellulaire applicable aux cellules de manière très diverse, reposant sur une technique de substitution d'eau distillée à utiliser lors de la préparation d'une suspension cellulaire d'eau de mer des grands fonds ou une technique de substitution de nutriants à ajouter à une suspension cellulaire d'eau de mer des grands fonds, ainsi qu'une méthode de sous-culture, dans laquelle des cellules humaines normales peuvent proliférer dans des conditions exemptes de sérum, selon la méthode de culture cellulaire susmentionnée.
PCT/JP2003/003941 2002-05-16 2003-03-28 Methode de culture cellulaire au moyen d'eau de mer des grands fonds WO2003097819A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003236182A AU2003236182A1 (en) 2002-05-16 2003-03-28 Cell culture method using marine deep water

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002-141036 2002-05-16
JP2002141036A JP2003334066A (ja) 2002-05-16 2002-05-16 海洋深層水を用いた細胞培養方法

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WO2003097819A1 true WO2003097819A1 (fr) 2003-11-27

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JP (1) JP2003334066A (fr)
AU (1) AU2003236182A1 (fr)
TW (1) TW200307046A (fr)
WO (1) WO2003097819A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2022004822A1 (fr) * 2020-06-30 2022-01-06 国立大学法人高知大学 Milieu de culture contenant de l'eau de mer profonde destiné à être utilisé dans la culture d'une cellule tissulaire dérivée d'un cellule sanguine d'appendice foetal et/ou de cordon ombilical

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002153267A (ja) * 2000-11-22 2002-05-28 Toyama Prefecture 深層水を利用した動物卵子及び初期胚の培養方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002153267A (ja) * 2000-11-22 2002-05-28 Toyama Prefecture 深層水を利用した動物卵子及び初期胚の培養方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Hironori MORIYAMA et al:, "Shoyu Jozo Biseibutsu no oyobosu Shinsosui no Eikyo", Reports of Kochi Prefectural Industrial Technology Center, Vol. 32, 19 December, 2001 (19.12.01), pages 103 to 105 *
Kenji YOTSUSHIMA et al:, "Baiyo Ekichu ni Tenka shita Kaiyo Shinsosui ga Ushi Taigai Yurai Hai no Hatsuiku ni Oyobosu Eikyo", Dai 93 kai The Japanese Society of Animal Reproduction Koen Yoshi, published on 15 September, 2000 (15.09.00), page 69, I-3-8 *
Takayuki HONMA: "Nettai Kaiiki oyobi Seitaibutsu ni okeru Shinsosui Riyo no Chosa Kenkyu Dai 3sho Seibutsu koka no Kenkyu II Seitaibutsu o Mochiita Saibo Kassei no Kenkyu 3 Sendoeki to no Daichokin to Kobo no Seiiku eno Eikyo, Nettai Kaiiki oyobi Seitaibutsu ni okeru Shinsosui Riyo no Chosa Kenkyu", Heisei 13 nendo Kenkyu Seika Hokokusho, April, 2001, pages 96 to 98 *

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JP2003334066A (ja) 2003-11-25
AU2003236182A1 (en) 2003-12-02
TW200307046A (en) 2003-12-01

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