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WO2018038032A1 - Récipient de culture annulaire, système de culture de cellules, et procédé de culture de cellules - Google Patents

Récipient de culture annulaire, système de culture de cellules, et procédé de culture de cellules Download PDF

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Publication number
WO2018038032A1
WO2018038032A1 PCT/JP2017/029723 JP2017029723W WO2018038032A1 WO 2018038032 A1 WO2018038032 A1 WO 2018038032A1 JP 2017029723 W JP2017029723 W JP 2017029723W WO 2018038032 A1 WO2018038032 A1 WO 2018038032A1
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WO
WIPO (PCT)
Prior art keywords
tube
annular
culture
cells
cell
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Ceased
Application number
PCT/JP2017/029723
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English (en)
Japanese (ja)
Inventor
郷史 田中
貴彦 戸谷
亮 末永
直己 高橋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toyo Seikan Group Holdings Ltd
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Toyo Seikan Group Holdings Ltd
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Filing date
Publication date
Application filed by Toyo Seikan Group Holdings Ltd filed Critical Toyo Seikan Group Holdings Ltd
Publication of WO2018038032A1 publication Critical patent/WO2018038032A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a cell culture method for culturing a large amount of cells in a closed container.
  • a culture container for culturing cells is manufactured by making a culture bag into a donut shape, vibrating on a rotary vibration table, and mixing the culture solution well in the bag. Proposed.
  • the present inventors have intensively studied, using an elongated tube formed using a flexible member, connecting the ends of the tube with a connecting member, and forming a sealed space in the formed tube.
  • the inventors have found that the above problems can be solved by culturing the cells while feeding the cells in one direction in a state where the cells and the culture solution are enclosed, and the present invention has been completed.
  • adherent cells and floating cells that can be cultured by forming aggregates are sealed together with the culture solution, and a pump is attached to drive this to drive the tube.
  • the cells can be cultured while feeding the culture medium and cells in one direction.
  • examples of the culture container formed using a tube include the continuous culture apparatus described in Patent Document 1 and the culture / recovery apparatus described in Patent Document 2.
  • the continuous culture apparatus described in Patent Document 1 cannot cultivate cells while feeding the culture solution and cells in the tube in one direction.
  • the culture / recovery device described in Patent Document 2 is a device in which cells are attached to the inner surface of the tube and cultured, and the cultured cells are detached and recovered by deforming the tube, and the cells are attached to the container. It was not possible to culture the cells while feeding the culture solution and the cells in the tube in one direction.
  • An object of the present invention is to provide an annular culture vessel, a cell culture system, and a cell culture method capable of obtaining a uniform aggregate and easily producing a culture vessel having a large capacity.
  • the annular culture container of the present invention comprises an elongated tube formed using a flexible member, and the tube ends are connected by one or two or more connecting members.
  • An annular sealed space in which cells are cultured is formed.
  • the cell culture system of the present invention includes the annular culture container and a peristaltic pump, and the pump is attached to the tube, and the culture medium and cells in the tube are driven by driving the pump. However, the culture is performed while being fed in one direction in the tube.
  • the cell culturing method of the present invention is a cell culturing method using the above cell culturing system, wherein an adherent cell or a floating cell that can be cultured by forming an aggregate is formed in the tube together with a culture solution, and the pump By driving the culture medium, the culture solution and cells in the tube are cultured while being fed in one direction in the tube.
  • the culture solution and cells in the tube can be efficiently fed in one direction, thereby preventing the cell aggregates from colliding with each other and obtaining a uniform aggregate.
  • a culture container having a large capacity can be easily produced.
  • FIG. 1 is a perspective view showing a schematic configuration of an annular culture vessel according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing the configuration of the annular culture vessel according to the embodiment of the present invention.
  • FIG. 3 is a schematic view showing a configuration of an annular culture vessel formed by connecting a plurality of annular spaces according to an embodiment of the present invention.
  • FIG. 4 shows a step of adding a culture solution using an annular culture vessel formed by connecting a plurality of annular spaces according to an embodiment of the present invention.
  • FIG. 5 shows a state in which the annular spaces of the annular culture vessel formed by connecting a plurality of annular spaces according to the embodiment of the present invention are stacked and arranged.
  • annular culture vessel the cell culture system, and the cell culture method of the present invention will be described in detail.
  • present invention is not limited to the specific contents of the following embodiments.
  • the annular culture container of this embodiment is composed of an elongated tube formed using a flexible member, and the end of the tube is connected by one or more connecting members to culture cells in the tube. A sealed space is formed.
  • the cell culture system of this embodiment includes such an annular culture container and a peristaltic pump, and the pump is attached to the tube. By driving the pump, the culture medium and cells in the tube are The culture is performed while being fed in one direction in the tube.
  • the cell culture method of the present embodiment is a cell culture method using such a cell culture system, and encloses adherent cells or floating cells that can be cultured by forming aggregates in a tube together with a culture solution, By driving the pump, the culture medium and the cells in the tube are cultured while being fed in one direction in the tube.
  • the annular culture container of the present embodiment has an elongated tube 1 made of a flexible member connected by a connecting member 11 to form an annular structure, and is sealed in the tube 1. A space is formed.
  • the term “annular” means that the tubes 1 are connected so as to form a ring, and are not limited to being connected in a circular shape, but are connected so as to form a rectangle or other shapes as shown in FIG. It may be.
  • the tube 1 in the annular culture container of the present embodiment may be formed by combining a plurality of tubes having different materials and rigidity using a connecting member or the like.
  • the tube 1 is preferably made of a gas permeable member.
  • the oxygen permeability coefficient is 400 ml ⁇ mm / m 2 ⁇ day ⁇ atm (37 ° C-80% RH) or more
  • the carbon dioxide permeability coefficient is 1200 ml ⁇ mm / m 2 ⁇ day ⁇ atm (37 ° C-80%).
  • RH or more is preferable
  • the oxygen permeability coefficient is 1000 ml ⁇ mm / m 2 ⁇ day ⁇ atm (37 ° C.-80% RH) or more
  • the carbon dioxide permeability coefficient is 3000 ml ⁇ mm / m 2 ⁇ day ⁇ atm. More preferably (37 ° C.-80% RH) or higher. If the tube 1 has such gas permeability, excellent cell growth efficiency can be obtained.
  • the tube 1 is partly or entirely made of a member having elastic deformability. Since at least a part of the tube 1 is elastically deformable, the culture medium and cells in the tube 1 can be efficiently sent using a peristaltic pump (also referred to as a tube pump, a roller pump, or a squeezing pump). can do. Moreover, since the tube 1 can be bent freely, the annular culture vessel can be arranged with excellent space efficiency. Furthermore, it is not necessary to use an air discharge means such as a pump by extracting the air from the annular culture vessel and filling the culture solution after being in a vacuum state, and the number of members can be reduced.
  • a peristaltic pump also referred to as a tube pump, a roller pump, or a squeezing pump.
  • an air discharge means such as a pump by extracting the air from the annular culture vessel and filling the culture solution after being in a vacuum state, and the number of members can be reduced.
  • the tube 1 has transparency so that the contents can be confirmed.
  • the material of the tube 1 include silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, Styrenic elastomers such as SBS (styrene / butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butylene / styrene), SEPS (styrene / ethylene / propylene / styrene), polyolefin resin, fluorine A resin or the like can be used. That is, it is preferable to use these as the material of the tube 1 and to have
  • the inner surface of the tube 1 is preferably a low adhesion surface.
  • the adherent cell etc. which can be cultured by forming an aggregate can be cultured suitably.
  • the low cell adhesion treatment can be performed on the inner surface of the tube 1 using, for example, MPC (2-Methacryloylethyl Phosphoryl Choline) polymer, hydrogel, or the like.
  • a size adjusting filter for adjusting the size of the cell aggregate in the tube 1.
  • the connecting member 11 forms an annular sealed space in the tube 1 by connecting the end portions of the tube 1.
  • a T-shaped tube connector is used as the connecting member 11, and the end portion of the tube 1 is inserted and connected to the facing opening.
  • vertical to the tube 1 is used as the port 12, and can be connected to an external bag etc. via another tube.
  • the connection member 11 is not limited to such a T-shaped tube connector, and may be a tube connector such as a Y-type, an I-type, or an X-type, or other tube connection device.
  • the tube 1 is provided with one or more ports 12 for taking in and out substances from the outside of the annular culture container.
  • This substance includes a liquid such as a culture solution, a gas such as air, and a solid matter such as a cell.
  • the port 12 is filled with a culture solution bag 5 for supplying the culture solution into the tube 1 or the air in the tube 1 to fill the tube 1 with the culture solution.
  • An air exhaust bag 6 or an air vent filter for pouring can be connected.
  • a sampling tube can also be connected to the port 12.
  • tube clamps are attached to the tubes connecting the tube 1 and the culture solution bag 5, and the tube 1 and the air exhaust bag 6, respectively.
  • the tube clamp between the tube 1 and the culture solution bag 5 is removed, the pump 4 is driven, and the culture solution in the culture solution bag 5 is filled into the tube 1.
  • the tube clamp between the tube 1 and the air exhaust bag 6 is removed, the pump 4 is driven, and the air in the tube 1 is discharged to the air exhaust bag 6.
  • a tube clamp is attached between the tube 1 and the air exhaust bag 6.
  • a tube clamp is attached between the tube 1 and the culture solution bag 5.
  • the sealed space in the tube 1 is filled with the cells 2 and the culture solution 3, and the cells 2 can be cultured while being fed in one direction in the tube 1.
  • the cell 2 is schematically represented largely as an aggregate.
  • the flow of the cells 2 and the culture solution 3 in the tube 1 can be performed by driving the pump 4.
  • a peristaltic pump that can send the cells 2 and the culture solution 3 in the tube 1 by squeezing the tube 1 can be suitably used.
  • an adherent cell or a floating cell that can be cultured by forming an aggregate can be preferably used.
  • adherent cells such as iPS cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells, and floating cells such as lymphocytes, monocytes, erythrocytes, and megakaryocytes. be able to.
  • adherent cells such as iPS cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells, and floating cells such as lymphocytes, monocytes, erythrocytes, and megakaryocytes.
  • the annular culture container of this embodiment can be easily manufactured by connecting the elongate tube 1 with the connection member 11, and can manufacture a culture container with a large capacity
  • the annular culture container of this embodiment is formed by connecting two or more tubes 1 with the connecting member 11 and connecting a plurality of annular spaces.
  • three annular spaces may be connected and formed, but the number of annular spaces is not limited to this, and is two. May be four or more.
  • annular space is connected to the cyclic
  • they are referred to as a first annular space, a second annular space, and a third annular space in order from the left.
  • the culture medium bag 5 is connected only to the first annular space, and the pump 4 is attached. However, both or one of them may be connected to or attached to the second annular space and / or the third annular space. it can.
  • Cell culture can be performed by, for example, the method shown in FIG. 4 using the annular culture vessel formed by connecting a plurality of annular spaces of the present embodiment.
  • the tube clamp attached to the connecting member 11 connecting the first annular space and the second annular space is removed, and the tube clamps of the culture medium bag 5 and the air exhaust bag 6 are removed, and the pump 4 is driven.
  • the tube 1 forming the first annular space is filled with the cells and the culture solution, and the cells are sent in one direction in the tube 1 to thereby perform the cell culture. Do.
  • the cells grow in the first annular space to a certain density, they are then attached to the tube clamp attached to the tube 1 of the second annular space and the air exhaust bag 6 of the second annular space. Remove the tube clamp and remove the tube clamp of the culture medium bag 5. Moreover, a tube clamp is attached to the connection member 11 that connects the first annular space and the second annular space, and the pump 4 is driven. Then, the cell 1 is formed by filling the tube 1 forming the first annular space and the second annular space with the cells and the culture solution and feeding them in one direction in the tube 1.
  • the tube clamp attached to the tube 1 in the third annular space and the air in the third annular space The tube clamp attached to the exhaust bag 6 is removed, and the tube clamp of the culture solution bag 5 is removed. Moreover, a tube clamp is attached to the connection member 11 that connects the second annular space and the third annular space, and the pump 4 is driven. Then, the cells 1 and the culture medium are filled in the tube 1 forming the first annular space, the second annular space, and the third annular space. Incubate. When there are four or more annular spaces, the same process is repeated. By culturing cells in this way using an annular culture vessel formed by connecting a plurality of annular spaces of this embodiment, the culture space is appropriately increased in accordance with the proliferation of the cells and a culture solution is added. The cells can be efficiently propagated.
  • the annular culture container formed by connecting a plurality of such annular spaces can be arranged by stacking the annular spaces on the support pillar 7 as shown in FIG.
  • the figure shows an annular culture vessel formed by connecting four annular spaces.
  • the annular culture container can be three-dimensionally arranged, and high space efficiency can be obtained. That is, by producing an annular culture vessel having a large number of annular spaces, it is possible to easily produce a culture vessel having a large capacity and excellent space efficiency.
  • the culture solution and the cells in the tube can be efficiently fed in one direction, whereby the cells
  • the agglomerates can be prevented from colliding with each other to obtain a uniform agglomerate, and a large-capacity culture vessel can be easily produced.
  • by producing an annular culture vessel having a large number of annular spaces it is possible to easily produce a culture vessel having a large capacity and excellent space efficiency.
  • the present invention can be suitably used for culturing a large amount of cells that can be cultured by forming aggregates.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
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  • Sustainable Development (AREA)
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  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un récipient de culture annulaire pourvu d'un tube allongé (1) formé à l'aide d'un élément souple, dans lequel un espace annulaire hermétiquement scellé pour la culture de cellules (2) est formé dans le tube par le raccordement des extrémités du tube (1) au moyen d'un ou plusieurs éléments de raccordement (11).
PCT/JP2017/029723 2016-08-22 2017-08-21 Récipient de culture annulaire, système de culture de cellules, et procédé de culture de cellules Ceased WO2018038032A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016161768A JP2018029488A (ja) 2016-08-22 2016-08-22 環状培養容器、細胞培養システム、及び細胞培養方法
JP2016-161768 2016-08-22

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WO2018038032A1 true WO2018038032A1 (fr) 2018-03-01

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521872A (zh) * 2021-06-24 2022-12-27 清华大学 一种管式培养设备

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109694825A (zh) * 2019-02-20 2019-04-30 湖南省肿瘤医院 一种培养3d细胞环的模具
JP7321438B2 (ja) * 2019-06-25 2023-08-07 株式会社アイカムス・ラボ 多流路平板ポンプ及び細胞培養装置
WO2020262354A1 (fr) * 2019-06-28 2020-12-30 アイ ピース， インコーポレイテッド Récipient de culture cellulaire et appareil de culture cellulaire
KR102096500B1 (ko) 2019-12-17 2020-04-02 주식회사 이뮤니스바이오 도넛형 세포 배양 백 및 이를 포함하는 세포 배양 시스템
KR20250114992A (ko) 2024-01-23 2025-07-30 경상국립대학교산학협력단 전단응력 적용이 가능한 트렌스웰 시스템용 세포 배양 디쉬 및 이를 포함하는 트렌스웰 시스템

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000287672A (ja) * 1999-04-06 2000-10-17 Canon Inc チューブ状培養槽による微生物の連続培養方法及び装置
JP2005087114A (ja) * 2003-09-18 2005-04-07 Hitachi Plant Eng & Constr Co Ltd 細胞の培養・回収方法及び装置
JP2007181410A (ja) * 2006-01-04 2007-07-19 Kagoshima Univ 中空穴あき体を用いる微生物の培養方法及びそのための装置
JP2007522825A (ja) * 2004-02-23 2007-08-16 クレシー，ウッド，フランソワ，マリ ド より適切な細胞変異体を選択可能な移動型容器を備えた連続培養装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000287672A (ja) * 1999-04-06 2000-10-17 Canon Inc チューブ状培養槽による微生物の連続培養方法及び装置
JP2005087114A (ja) * 2003-09-18 2005-04-07 Hitachi Plant Eng & Constr Co Ltd 細胞の培養・回収方法及び装置
JP2007522825A (ja) * 2004-02-23 2007-08-16 クレシー，ウッド，フランソワ，マリ ド より適切な細胞変異体を選択可能な移動型容器を備えた連続培養装置
JP2007181410A (ja) * 2006-01-04 2007-07-19 Kagoshima Univ 中空穴あき体を用いる微生物の培養方法及びそのための装置

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521872A (zh) * 2021-06-24 2022-12-27 清华大学 一种管式培养设备

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