WO2017170335A1 - Cuve de culture cellulaire, gabarit de support pour cuve de culture cellulaire et procédé de culture cellulaire - Google Patents

Cuve de culture cellulaire, gabarit de support pour cuve de culture cellulaire et procédé de culture cellulaire Download PDF

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Publication number
WO2017170335A1
WO2017170335A1 PCT/JP2017/012265 JP2017012265W WO2017170335A1 WO 2017170335 A1 WO2017170335 A1 WO 2017170335A1 JP 2017012265 W JP2017012265 W JP 2017012265W WO 2017170335 A1 WO2017170335 A1 WO 2017170335A1
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WO
WIPO (PCT)
Prior art keywords
cell culture
cells
recess
container
container according
Prior art date
Application number
PCT/JP2017/012265
Other languages
English (en)
Japanese (ja)
Inventor
郷史 田中
亮 末永
貴彦 戸谷
Original Assignee
東洋製罐グループホールディングス株式会社
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.)
Filing date
Publication date
Priority claimed from JP2016208335A external-priority patent/JP2017184716A/ja
Application filed by 東洋製罐グループホールディングス株式会社 filed Critical 東洋製罐グループホールディングス株式会社
Priority to KR1020207035625A priority Critical patent/KR102328979B1/ko
Priority to KR1020187022886A priority patent/KR102222874B1/ko
Priority to CN201780011599.0A priority patent/CN108699500B/zh
Priority to EP17774872.0A priority patent/EP3438237A4/fr
Publication of WO2017170335A1 publication Critical patent/WO2017170335A1/fr
Priority to US16/148,229 priority patent/US11414637B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means

Definitions

  • the present invention relates to a cell culture vessel, a cell culture vessel support jig, and a cell culture method for efficiently culturing various cells.
  • well plates and flasks are often used as culture vessels for subculture.
  • a well plate in order to obtain an appropriate cell density, add cells to the individual wells together with the culture medium, start culturing, allow the cells to grow sufficiently in the well, and then transfer to the flask. Incubate with additional culture medium according to the growth, and when it grows to a certain volume, transfer to a flask with a larger volume and add more culture medium. It is known (see, for example, paragraph [0027] of Patent Document 1).
  • Patent Document 2 proposes a flask-type culture vessel in which a plurality of recesses are formed on one surface of a polyhedral shape such as a rectangular parallelepiped as a flask-type culture vessel.
  • an agglomerate formed by coalescence of single cells is formed in a recess that is a plurality of first culture portions provided in the first culture surface, and this is formed on the second culture surface in the container. It moves to the 2nd culture part with a larger area than a culture part, and is trying to become a bigger aggregate.
  • An object of the present invention is to provide a cell culture container, a cell culture container support jig, and a cell culture method.
  • the cell culture container according to the present invention includes a container main body made of a gas permeable plastic film and an injection port, and the container main body has a peripheral portion sealed and a container top surface side having a bulging shape.
  • a plurality of recesses to be cell culture parts are provided on the bottom surface of the container body.
  • the cell culture container support jig is a jig for supporting the cell culture container, and is configured to support the bottom side of the container body in a non-contact manner with the recess.
  • the cell culture method according to the present invention is a cell culture method using the above-described cell culture container, wherein the bottom surface side of the container body is supported in a non-contact manner on the concave portion, and the cell is formed on the bottom portion of the concave portion.
  • cells can be efficiently cultured in the same container while reducing the risk of contamination while maintaining an appropriate cell density during culture.
  • a cell culture container 1 shown in FIG. 1 includes a container main body 2 made of a gas permeable plastic film and an injection port 3 made of a tubular member through which a culture medium, cells, and the like can circulate.
  • the container body 2 has a bulging shape in which the peripheral portion is sealed and the top surface 2a side bulges like a plateau, and the edge of the flat top surface 2a is inclined so as to be connected to the peripheral portion. Is formed.
  • the bottom surface 2b of the container body 2 is provided with a plurality of recesses 4 serving as cell culture parts.
  • the concave portion 4 provided on the bottom surface 2b of the container body 2 has an opening diameter (diameter) D of 0 in order to suppress the movement of cells in the container body 2 so that cells in culture remain in the single concave portion 4. It is preferably 3 to 10 mm, more preferably 0.3 to 5 mm, still more preferably 0.5 to 4 mm, particularly preferably 0.5 to 2 mm, and the depth d is 0.1 mm or more. Is preferred.
  • the opening diameter of the recess 4 may be the same for all the recesses 4. For example, the bottom surface 2 b is divided into a plurality of regions, and the opening diameter of the recess 4 is made different for each region.
  • the recessed part 4 to be provided may include two or more kinds of recessed parts having different opening diameters.
  • the top surface 2a side of the container body 2 is bulged in a plateau shape, and the edge of the top surface 2a is inclined so as to be connected to the peripheral portion.
  • the amount of the medium containing the cells decreases on the peripheral side and settles in the medium.
  • the number of cells is also reduced.
  • the opening diameters of all the recesses 4 are the same, the number of cells that settle into the recesses 4 on the peripheral side is reduced, so the same number of cells settle in all the recesses 4.
  • the shape of the recess 4 is a spherical crown so that cells can easily gather at the bottom of the recess 4, but the shape of the recess 4 is not limited to this.
  • the ratio d / D of the depth d to the opening diameter D of the recess 4 is preferably set to 0.05 to 1.
  • the occupied area of the concave portion 4 occupying the bottom surface 2b is preferably as large as possible within a range in which the moldability is not impaired in order to avoid cells from staying in the portion other than the concave portion 4 of the bottom surface 2b. Specifically, it is preferably 30 to 90% with respect to the area of the bottom surface 2b.
  • the recesses 4 are preferably arranged in a zigzag pattern as shown in the figure so that the area occupied by the recesses 4 in the bottom surface 2b is as large as possible. However, the recesses 4 may be arranged in a lattice pattern as necessary.
  • the size of the container body 2 is not particularly limited, but is preferably, for example, 50 to 500 mm in length and 50 to 500 mm in width.
  • Such a cell culture container 1 can be manufactured as follows, for example. First, a top surface side plastic film that becomes the top surface 2 a side of the container body 2 and a bottom surface side plastic film that becomes the bottom surface 2 b side of the container body 2 are prepared. And the top surface side plastic film is shape
  • the top side plastic film and the bottom side plastic film molded as described above are overlapped, and the peripheral part is heat-sealed with a tubular member forming the injection port 3 at a predetermined position. And then trim the periphery if necessary. Thereby, the cell culture container 1 as shown in FIG. 1 can be manufactured.
  • the gas permeability of the plastic film forming the container body 2 is determined according to the gas permeability test method of JIS K 7126.
  • the oxygen permeability measured at a test temperature of 37 ° C. is 5000 mL / (m 2 ⁇ day ⁇ atm. ) Or more.
  • it is preferable that a part or all of the plastic film has transparency so that the progress of cell culture and the state of cells can be confirmed.
  • the material used for the plastic film forming the container body 2 is not particularly limited as long as it has a desired gas permeability.
  • examples thereof include thermoplastic resins such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone elastomer, polystyrene elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP). These may be used as a single layer, or may be used by laminating the same or different materials, but has a layer that functions as a sealant layer in consideration of heat fusion when sealing the periphery. Is preferred.
  • the plastic film used for forming the container body 2 so as to have an appropriate shape retaining property capable of maintaining the bulging shape on the top surface 2a side and the shape of the concave portion 4 while having flexibility.
  • the thickness is preferably 30 to 200 ⁇ m.
  • the injection port 3 is composed of a tubular member through which a culture medium, cells, and the like can circulate, as described above, but the tubular member forming the injection port 3 is, for example, polyethylene, polypropylene, It can be molded into a predetermined shape by injection molding, extrusion molding, or the like using a thermoplastic resin such as vinyl chloride, polystyrene-based elastomer, or FEP.
  • a thermoplastic resin such as vinyl chloride, polystyrene-based elastomer, or FEP.
  • a port blocking prevention piece can be provided.
  • a port blockage prevention piece is provided, it is preferably provided so as to be positioned on the top surface 2a side of the container body 2 so as not to hinder the sedimentation of cells into the recess 4 provided on the bottom surface 2b.
  • the cells to be cultured together with the culture medium are placed in the vessel body 2 while maintaining a closed system via a liquid feeding tube connected to the injection port 3. inject. Then, the cells injected into the container body 2 settle in the medium and are collected at the bottom of each recess 4.
  • the bottom surface is deformed so that the periphery is lifted as the container is filled with the content liquid.
  • the top surface 2a side of the container body 2 has a bulging shape, and by designing the bulging shape on the top surface 2a side in consideration of the injection amount, It is possible to suppress the deformation of the bottom surface 2b when the cells to be cultured are injected together with the medium. Accordingly, the cells can be uniformly accommodated in the respective recesses 4 without the recesses 4 provided on the bottom surface 2b being inclined.
  • a cell low adhesion treatment is applied to the bottom surface 2b including the recesses 4 so that the cells settled in the medium are likely to collect at the bottoms of the recesses 4 so that the cells do not adhere easily.
  • the low cell adhesion treatment include, for example, a treatment for imparting hydrophilicity to the surface of the plastic film by a surface treatment such as a plasma treatment, a phospholipid polymer, a surfactant, a protein such as albumin, and the like. Treatment for inhibiting the adsorption of cell adhesion protein.
  • the bottom surface 2b side also has a bulging shape that bulges in a plateau like the top surface 2a side. By doing in this way, it is formed so that the edge of bottom face 2b may incline and continue to a peripheral part, and it can avoid that a cell retains in the peripheral part side of bottom face 2b. Furthermore, it is preferable that the bottom surface 2b side of the container body 2 also has a bulging shape in order to suppress deformation of the bottom surface 2b when the cells to be cultured are injected together with the culture medium.
  • the cells injected into the container body 2 settle in the medium and gather at the bottom of each recess 4, and the cell density is increased in a state where the cell density is increased. It can induce culture and differentiation well. And since the container main body 2 consists of a plastic film which has gas permeability, it can supply sufficient quantity of oxygen to the cell in culture
  • the concave portion 4 is formed on the bottom surface side plastic film as described above, the plastic film is stretched so that the thickness of the concave portion 4 is other than the concave portion 4 of the bottom surface 2b. It is preferable to make the thickness thinner than the thickness of the portion, whereby the gas permeability of the recess 4 can be increased, and sufficient oxygen can be supplied to the cells collected in the recess 4.
  • cells when performing cell culture using the cell culture container 1, cells can be cultured under predetermined conditions in a culture vessel such as a CO 2 incubator.
  • the cell culture vessel 1 is preferably installed on a frame in the incubator via a jig 10 that can support the bottom surface 2 b side of the concave portion 4 in a non-contact manner.
  • the concave portion 4 is surely prevented from being crushed and deformed, and the cells in culture collected in the concave portion 4 are prevented from flowing out around the concave portion 4.
  • the gas permeability from the bottom surface 2b can be maximized.
  • a perforation or a recess having a diameter that allows the recess 4 to be inserted in a non-contact manner corresponds to the arrangement of the recess 4
  • the top surface 2a is used as the culture surface with the top of the cell culture container 1 reversed. By using it, the culture area can be expanded. This makes it possible to continue cell culture while maintaining an appropriate cell density in the same container.
  • the cells to be cultured are anchorage-dependent cells
  • an enzyme solution for peeling the cells from the bottom surface of the recesses 4 is added as necessary. In this case, the enzyme solution can be injected into the container body 2 while maintaining a closed system via the liquid feed tube connected to the injection port 3. .
  • cell culture usually requires a period of several days to several weeks, and during that period, it is necessary to add a medium or change a medium as necessary. These operations can be easily performed while maintaining the closed system by adding the medium or replacing the medium through the liquid feeding tube connected to the. Furthermore, after completion of the culture, the cells in the cell culture vessel 1 can be recovered while maintaining the closed system via the liquid feeding tube connected to the injection port 3.
  • the cell density at the time of culture is appropriately maintained while maintaining a closed system without subculture, and the cells are efficiently used. It is possible to multiply it. Moreover, a complicated transfer operation is not required, and cells can be efficiently cultured in the same container while reducing the risk of contamination.
  • the container body 2 is formed using a plastic film, it is lightweight even if the capacity is increased and is not bulky. Therefore, the container body 2 is suitable for large-scale cell culture. A sufficient amount of medium can be injected. In addition, the size of the container body 2 can be adjusted using a clamp or the like, and can be flexibly handled according to the number of cells and the amount of medium. On the other hand, in the flask type culture container as in Patent Document 2, it is necessary to replace the container with a different size.
  • the container main body 2 has a rectangular shape and includes the injecting port 3 on one side of the short side, but is not limited thereto.
  • the shape of the container body 2 may be a square shape, an oval shape, a circular shape, or the like, and can be various shapes as necessary.
  • the position and the number of the injection ports 3 can be changed as appropriate.
  • the present invention can be used as a technique for efficiently culturing various cells.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
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  • Sustainable Development (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention décrit une cuve de culture cellulaire (1) prévue avec : un corps principal de cuve (2) comprenant des films plastiques perméables aux gaz ; et un orifice d'injection/évacuation(3). Le corps principal de cuve (2) est hermétiquement scellé au niveau de sa partie périphérique, présente une forme bombée sur le côté face supérieure (2a) et est prévu avec une pluralité de renfoncements (4) servant de parties de culture cellulaire sur la face inférieure (2b). Dans la cuve de culture cellulaire (1), les cellules à cultiver sont injectées avec un milieu de culture dans le corps principal de cuve (2) à travers l'orifice d'injection/évacuation (3), et cultivées. Ceci permet aux cellules d'être efficacement cultivées dans la même cuve tout en conservant une densité appropriée des cellules lors de la culture et en réduisant le risque de contamination.
PCT/JP2017/012265 2016-03-31 2017-03-27 Cuve de culture cellulaire, gabarit de support pour cuve de culture cellulaire et procédé de culture cellulaire WO2017170335A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
KR1020207035625A KR102328979B1 (ko) 2016-03-31 2017-03-27 세포 배양 용기, 세포 배양 용기의 지지 지그 및 세포 배양 방법
KR1020187022886A KR102222874B1 (ko) 2016-03-31 2017-03-27 세포 배양 용기, 세포 배양 용기의 지지 지그 및 세포 배양 방법
CN201780011599.0A CN108699500B (zh) 2016-03-31 2017-03-27 细胞培养容器、细胞培养容器的支撑夹具、以及细胞培养方法
EP17774872.0A EP3438237A4 (fr) 2016-03-31 2017-03-27 Cuve de culture cellulaire, gabarit de support pour cuve de culture cellulaire et procédé de culture cellulaire
US16/148,229 US11414637B2 (en) 2016-03-31 2018-10-01 Cell culture vessel, support jig for cell culture vessel and cell culture method

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2016-070718 2016-03-31
JP2016070718 2016-03-31
JP2016-208335 2016-10-25
JP2016208335A JP2017184716A (ja) 2016-03-31 2016-10-25 細胞培養容器、細胞培養容器の支持治具、及び細胞培養方法

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US16/148,229 Continuation US11414637B2 (en) 2016-03-31 2018-10-01 Cell culture vessel, support jig for cell culture vessel and cell culture method

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018230544A1 (fr) * 2017-06-15 2018-12-20 東洋製罐グループホールディングス株式会社 Procédé et dispositif de culture cellulaire
WO2020100567A1 (fr) * 2018-11-17 2020-05-22 東洋製罐グループホールディングス株式会社 Solution de remplissage de milieu, procédé de remplissage de milieu, récipient de culture et appareil d'élimination de bulles pour support de remplissage
CN111511898A (zh) * 2018-01-09 2020-08-07 东洋制罐集团控股株式会社 细胞培养方法及装置
WO2020166233A1 (fr) * 2019-02-12 2020-08-20 東洋製罐グループホールディングス株式会社 Mécanisme de régulation de densité de liquide, dispositif de culture et procédé de régulation de densité de liquide
WO2022014436A1 (fr) * 2020-07-11 2022-01-20 東洋製罐グループホールディングス株式会社 Récipient de culture cellulaire, procédé de fabrication de récipient de culture cellulaire, procédé de production de cellule, dispositif de culture cellulaire et gabarit de culture cellulaire

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JP2006055069A (ja) * 2004-08-20 2006-03-02 National Institute Of Advanced Industrial & Technology 細胞培養容器及び培養方法
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018230544A1 (fr) * 2017-06-15 2018-12-20 東洋製罐グループホールディングス株式会社 Procédé et dispositif de culture cellulaire
EP3640322A4 (fr) * 2017-06-15 2021-03-03 Toyo Seikan Group Holdings, Ltd. Procédé et dispositif de culture cellulaire
CN111511898A (zh) * 2018-01-09 2020-08-07 东洋制罐集团控股株式会社 细胞培养方法及装置
EP3739037A4 (fr) * 2018-01-09 2021-10-06 Toyo Seikan Group Holdings, Ltd. Procédé et dispositif de culture cellulaire
CN111511898B (zh) * 2018-01-09 2024-01-02 东洋制罐集团控股株式会社 细胞培养方法及装置
WO2020100567A1 (fr) * 2018-11-17 2020-05-22 東洋製罐グループホールディングス株式会社 Solution de remplissage de milieu, procédé de remplissage de milieu, récipient de culture et appareil d'élimination de bulles pour support de remplissage
JP2020080670A (ja) * 2018-11-17 2020-06-04 東洋製罐グループホールディングス株式会社 培地充填液、培地充填方法、培養容器、及び培地充填用気泡除去装置
JP7322384B2 (ja) 2018-11-17 2023-08-08 東洋製罐グループホールディングス株式会社 培地充填液、培地充填方法、培養容器、及び培地充填用気泡除去装置
WO2020166233A1 (fr) * 2019-02-12 2020-08-20 東洋製罐グループホールディングス株式会社 Mécanisme de régulation de densité de liquide, dispositif de culture et procédé de régulation de densité de liquide
JP2020127391A (ja) * 2019-02-12 2020-08-27 東洋製罐グループホールディングス株式会社 液厚制御機構、培養装置、及び液厚制御方法
JP7379824B2 (ja) 2019-02-12 2023-11-15 東洋製罐グループホールディングス株式会社 液厚制御機構、培養装置、及び液厚制御方法
WO2022014436A1 (fr) * 2020-07-11 2022-01-20 東洋製罐グループホールディングス株式会社 Récipient de culture cellulaire, procédé de fabrication de récipient de culture cellulaire, procédé de production de cellule, dispositif de culture cellulaire et gabarit de culture cellulaire

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