WO2016117281A1 - Récipient de culture cellulaire, procédé de culture cellulaire et procédé d'utilisation du récipient de culture cellulaire - Google Patents

Récipient de culture cellulaire, procédé de culture cellulaire et procédé d'utilisation du récipient de culture cellulaire Download PDF

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Publication number
WO2016117281A1
WO2016117281A1 PCT/JP2016/000007 JP2016000007W WO2016117281A1 WO 2016117281 A1 WO2016117281 A1 WO 2016117281A1 JP 2016000007 W JP2016000007 W JP 2016000007W WO 2016117281 A1 WO2016117281 A1 WO 2016117281A1
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WO
WIPO (PCT)
Prior art keywords
cell culture
culture container
cell
frame
cells
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PCT/JP2016/000007
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English (en)
Japanese (ja)
Inventor
郷史 田中
庸一 石▲崎▼
聖真 田中
健一 森井
誠一 横尾
Original Assignee
東洋製罐グループホールディングス株式会社
株式会社ジェイ・エム・エス
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Application filed by 東洋製罐グループホールディングス株式会社, 株式会社ジェイ・エム・エス filed Critical 東洋製罐グループホールディングス株式会社
Publication of WO2016117281A1 publication Critical patent/WO2016117281A1/fr

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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 technique, and more particularly to a cell culture container, a cell culture method, and a method of using the cell culture container for culturing adherent cells.
  • cell culture is performed using a base material (scaffold) for adhering cells to produce a sheet or tissue.
  • a base material sinaffold
  • an open-type container having a large opening such as a petri dish
  • CPC cell processing center
  • FIG. 4 of Patent Document 1 describes a cell culture container in which a closed culture chamber is formed by a frame, a gas permeable membrane, and a surface facing the frame.
  • 1 and 2 of Patent Document 2 describe a cell culture container in which a closed space is formed by a container body having an opening and a gas-permeable film that seals the opening. According to these cell culture containers, it is possible to cultivate cells by attaching them on a gas permeable membrane or film in a closed system space, and it is possible to peel and collect the cell sheet from the gas permeable membrane or film. It has become.
  • these conventional cell culture containers have a problem that they are not suitable for work in a medical field because they are intended to peel and collect a cell sheet from a gas permeable membrane or film.
  • the cells to be cultured are corneal cells
  • the conventional cell culture container requires a post-treatment step such as culturing by attaching cells on a gas permeable membrane or film, and treating with a solution such as trypsin in order to separate the cells from the film. Complicated work was required. For this reason, it is not suitable for use in a medical field such as an operating room, and depending on the cultured cells, the cells may be damaged in the post-treatment process.
  • the present inventors have intensively studied, and can perform aseptic operations such as medium exchange while maintaining a closed system, and can easily take out the cultured cell sheets, tissue, etc. from the sealed space
  • a cell culture vessel was devised. If such a cell culture container is used, it becomes possible to culture cell sheets, tissue-like materials and the like at low cost without using a large-scale facility such as a cell processing center.
  • the present invention has been made in view of the above circumstances, and can perform cell culture in a closed system, and can easily take out a cultured cell sheet, tissue, etc., a cell culture container, a cell culture method, and a cell It aims at providing the usage method of a culture container.
  • an internal space is formed by a frame and upper and lower surfaces bonded to the front and back of the frame, and at least one of the upper and lower surfaces is a gas permeable film.
  • a base material for culturing adhesive cells in the internal space, and the frame body having at least one access port.
  • the cell culture method of the present invention is a method of culturing the adherent cells by enclosing a medium in such a cell culture container and seeding the adherent cells on the substrate through an access port.
  • the method for using the cell culture container of the present invention is a method for culturing adhesive cells using such a cell culture container and transplanting the adhesive cells together with the base material into a living body. This is a method of collecting the adhered base material.
  • a cell culture container a cell culture method, and a method for using the cell culture container, which can perform cell culture in a closed system and can easily take out the cultured cell sheet or tissue. It becomes possible.
  • FIG. 1 It is a perspective view which shows the cell culture container which concerns on 1st embodiment of this invention. It is the top view and front view which show the cell culture container which concerns on 1st embodiment of this invention. It is a figure which shows the state which took out the holding member provided with the base material from the cell culture container which concerns on 1st embodiment of this invention. It is a perspective view which shows the cell culture container which concerns on 2nd embodiment of this invention. It is a perspective view which shows the cell culture container which concerns on 3rd embodiment of this invention.
  • FIG. 1 is a perspective view showing a cell culture container of the present embodiment
  • FIG. 2 is a plan view and a front view showing the cell culture container of the present embodiment
  • FIG. 3 is a figure which shows the state which took out the holding member provided with the base material from the cell culture container of this embodiment.
  • the cell culture container 10 of the present embodiment has a frame 11 and an upper surface 14 and a lower surface 15 bonded to the front and back of the frame 11, and an internal space is formed by these. ing.
  • the frame 11 is a frame composed of hard side walls. Although it does not specifically limit as a material of this frame 11, From the viewpoint of adhere
  • the shape of the frame 11 is not particularly limited as long as the internal space can be formed.
  • the frame 11 can have a substantially rectangular parallelepiped shape as shown in FIGS. Further, it may be a cylindrical shape or a polygonal column shape.
  • At least one of the upper surface 14 and the lower surface 15 is made of a gas permeable film.
  • the gas permeable film has a gas permeability necessary for cell culture, and specifically has an oxygen permeability of 5000 ml / m 2 ⁇ day ⁇ atm (37 ° C.-80% RH) or more.
  • this gas permeable film preferably has low cytotoxicity, low elution properties, and suitability for radiation sterilization in order to achieve high cell growth efficiency.
  • a transparent gas permeable film in order to make it possible to observe the cultured cells from the outside of the cell culture vessel 10.
  • both the upper surface 14 and the lower surface 15 are comprised with a gas permeable film, while being able to improve gas permeability more, not only observation from an up-and-down surface is attained, but the detailed observation by a microscope is also attained. Therefore, it is preferable.
  • the material for the gas permeable film it is preferable to use a polyethylene resin.
  • the polyethylene resin include polyethylene, ethylene / ⁇ -olefin copolymer, ethylene / vinyl acetate copolymer, ionomer using ethylene / acrylic acid / methacrylic acid copolymer and metal ion, and the like. it can. Further, polyolefin, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, silicone resin and the like can also be used.
  • the easy peeling part used as a handle can be formed on one or both of the upper surface 14 and the lower surface 15.
  • an easy peel portion 141 is provided on the upper surface 14.
  • the upper surface 14 and / or the lower surface 15 on which the easy peeling portion is formed are bonded to the frame body 11 so as to have a relatively weak adhesive force so as to be easily peeled off.
  • the cell culture container 10 of this embodiment is equipped with the base material 12 for culture
  • the cells to be cultured are not particularly limited as long as they are adherent cells.
  • skin cells, corneal epithelial cells, corneal endothelial cells, vascular endothelial cells, epidermal cells, mesenchymal stem cells, chondrocytes and the like are preferably used. it can.
  • the base material 12 is used as a scaffold for the cells to adhere and proliferate when culturing adhesive cells.
  • the base material 12 can select an appropriate thing according to the cell to culture, For example, amniotic membrane, collagen, etc. can be used suitably. By using amniotic membrane as the base material 12, rejection can be made difficult to occur during transplantation.
  • the holding member 13 is a member that stably supports the base material 12 in the cell culture container 10.
  • a transwell is shown in FIG.
  • the transwell has a bottom surface formed of a gas permeable membrane, and the base material 12 is disposed on the bottom surface.
  • the transwell includes a hole 131 through which the medium can move in the cell culture container 10. For this reason, it is possible to stably arrange the holding member 13 in the cell culture container 10 as compared with the case where there is no hole 131.
  • the transwell has a substantially cylindrical shape at the bottom and a substantially truncated cone shape at the top, so that cultured cells can be formed in a circular shape.
  • the holding member 13 may be any member as long as it can hold the substrate 12, and other members such as a dish may be used in addition to the transwell.
  • the cell culture container 10 of this embodiment includes at least one access port in the frame 11.
  • the number is not particularly limited, and two or more may be provided.
  • three access ports are provided, one as a cell injection access port 111 and the other two as a medium transfer access port 112.
  • the cell injection access port 111 is made of an elastic body such as rubber or silicone, and a cell suspension containing cells is injected into the cell culture container 10 through the cell injection access port 111 using a syringe or the like. Can do.
  • tubes 16 are connected to the two medium transfer access ports 112, respectively, and one is used for adding cells and the other is used for collecting cells via the tubes 16.
  • the medium can be quickly exchanged inside.
  • a sample port and other ports may be provided, and a plurality of various ports may be provided.
  • an air vent port using a membrane filter is provided on the upper side of the frame body 11, when the culture medium is transferred into the cell culture container 10 at the start of culture, the operation of transferring the air in the container to the waste liquid container is not performed. In both cases, since air escapes through the air vent port, the culture can be started by a simpler operation.
  • the cell culture container 10 of this embodiment is provided with only one access port, all the movements related to the contents of the cell culture container 10 such as cell injection, medium transfer, and sampling are performed by one access port.
  • the adherent cells can be cultured in a closed system, and the cultured cells can be easily and safely collected.
  • the medium is sealed in the cell culture container 10 described above, and the adherent cells are seeded on the base material 12 via the access port. Culture is performed. Specifically, for example, it can be used in the following environment.
  • the cell culture container 10 of the present embodiment is taken out from the sterilization bag, the end of one tube 16 is connected to a medium supply container, and the end of the other tube 16 is connected to a waste liquid container. Connect to. Thereby, the internal space of the cell culture container 10 is maintained in a closed system.
  • the whole of the cell culture container 10, the medium supply container, and the waste liquid container may be referred to as a cell culture system.
  • the patient is operated to collect corneal epithelial cells, which are added to the medium and stirred to prepare a cell suspension.
  • this cell suspension is injected onto the substrate 12 from the cell injection access port 111 using a syringe. Further, the medium is supplied from the medium supply container into the cell culture container 10 via the tube 16 and the medium transfer access port 112. Thereby, cell culture is started in the cell culture vessel 10.
  • the cell culture can be continued aseptically even when the cell culture system is carried from the operating room to a non-sterile environment.
  • the cell culture system is again carried to the operating room in a sterile environment, and the easy peeling portion 141 of the cell culture container 10 is picked and the upper surface 14 is peeled off as shown in FIG.
  • the holding member 13 is taken out from the frame body 11.
  • the base material 12 is collect
  • the adherent cells can be cultured while maintaining a closed system without using a large-scale facility such as a cell processing center, and the substrate 12 to which the adherent cells are attached can be efficiently used. And can be quickly transplanted into a patient. For this reason, according to the cell culture method of this embodiment and the usage method of a cell culture container, an adhesive cell can be cultured at low cost and it can be used suitably for regenerative medicine.
  • FIG. 4 is a perspective view showing the cell culture container of the present embodiment.
  • the cell culture container 10 a of this embodiment has a lower surface 15 made of a gas permeable film, and a leg member 17 provided on the bottom of the frame body 11 so that gas can pass through the lower surface 15. This is different from the first embodiment. About another point, it can be set as the structure similar to 1st embodiment.
  • the cell culture container 10a is provided with leg members 17 at the four corners of the frame body 11, so that even when the cell culture container 10a is placed on a flat surface, the lower surface 15 is placed between the flat surface. A space can be provided. For this reason, according to the cell culture container 10a of this embodiment, it is possible to allow gas to permeate into the cell culture container 10a through this space and the lower surface 15.
  • the number, position, shape, and the like of the leg members 17 provided in the cell culture container 10a are not particularly limited as long as the cell culture container 10a can be stably placed. Further, the material of the cell culture vessel 10a is not particularly limited, but the same material as that of the frame 11 can be suitably used.
  • the cell culture container 10a of this embodiment by providing the leg member 17, the gas exchange efficiency in the cell culture container 10a can be improved, and the culture efficiency can be improved. Moreover, the cell culture container 10a of this embodiment can be applied to the same cell culture method and the usage method of a cell culture container as 1st embodiment.
  • FIG. 5 is a perspective view showing the cell culture container of the present embodiment.
  • the cell culture container 10b of the present embodiment is not provided with the holding member 13, but is provided with a base material 12a made of sponge as a scaffold for culturing adhesive cells. It is different from the form. About another point, it can be set as the structure similar to 1st embodiment.
  • the sponge is not particularly limited as long as it can be used as a scaffold for adhesive cells.
  • a sponge containing collagen can be preferably used.
  • a gel containing collagen a hydrogel using alginic acid or the like, a biological membrane such as amniotic membrane, or the like can be used.
  • the base material 12 can be directly arrange
  • the cell culture container 10b of the present embodiment can be applied to the same cell culture method as in the first embodiment and the method of using the cell culture container.
  • the present invention is not limited to the above embodiment, and that various modifications can be made within the scope of the present invention.
  • a plurality of cell culture vessels are provided, and a plurality of cultures can be performed simultaneously, or a sponge or a gel is used instead of the amniotic membrane in the holding member by combining the first embodiment and the third embodiment.
  • a sponge or a gel is used instead of the amniotic membrane in the holding member by combining the first embodiment and the third embodiment. It is possible to appropriately change the arrangement, for example, by providing a leg member in a cell culture container in which a sponge or gel is arranged without providing a holding member by combining the second embodiment and the third embodiment.
  • the present invention can be suitably used when culturing adherent cells simply and at low cost.

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Abstract

L'invention concerne : un récipient de culture cellulaire qui permet de réaliser une culture cellulaire dans un système fermé et d'enlever facilement une feuille de cellules cultivées, un matériau de type tissu cultivé ou analogue; un procédé de culture cellulaire; et un procédé d'utilisation du récipient de culture cellulaire. Un récipient de culture cellulaire selon l'invention est pourvu de : un cadre, un espace intérieur étant formé par une surface supérieure et une surface inférieure reliées de face et à l'arrière du cadre et au moins l'une parmi la surface supérieure et la surface inférieure comprenant un film perméable aux gaz; et un matériau de base pour la culture de cellules adhésives à l'intérieur de l'espace intérieur, le cadre étant muni d'au moins un orifice d'accès. Un milieu est scellé en son sein, des cellules adhésives sont semées sur le matériau de base via l'orifice d'accès et les cellules adhésives sont cultivées. Le matériau de base auquel les cellules adhésives sont attachées est également récupéré de manière à transplanter les cellules adhésives conjointement avec le matériau de base dans un corps vivant.
PCT/JP2016/000007 2015-01-21 2016-01-04 Récipient de culture cellulaire, procédé de culture cellulaire et procédé d'utilisation du récipient de culture cellulaire WO2016117281A1 (fr)

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JP2015-009374 2015-01-21
JP2015009374A JP2018033318A (ja) 2015-01-21 2015-01-21 細胞培養容器、細胞培養方法、及び細胞培養容器の使用方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020505030A (ja) * 2017-01-16 2020-02-20 レアクト４ライフ エッセエッレエッレ バイオリアクタおよびこのようなバイオリアクタの使用方法
CN112300929A (zh) * 2019-07-31 2021-02-02 上海新微技术研发中心有限公司 一种微流控实验板及双面细胞培养方法

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Publication number Priority date Publication date Assignee Title
CA2989768A1 (fr) * 2015-06-25 2016-12-29 Auckland Uniservices Limited Appareil comprenant un materiau permeable au gaz et methodes de croissance de cellules et de tissus epidermiques
JPWO2020195458A1 (ja) * 2019-03-28 2021-10-14 富士フイルム株式会社 測定装置、測定装置の作動プログラム及び測定方法
JP7310258B2 (ja) * 2019-04-19 2023-07-19 大日本印刷株式会社 生体物質用容器、生体物質用容器キット及び器具入り容器
JP7286749B1 (ja) 2021-12-23 2023-06-05 財團法人工業技術研究院 細胞及び組織シート形成パッケージ並びに細胞注入装置

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WO2012008368A1 (fr) * 2010-07-16 2012-01-19 株式会社日立製作所 Récipient de culture cellulaire et dispositif de culture cellulaire

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JP2006320304A (ja) * 2005-05-18 2006-11-30 Cellseed Inc 密閉系細胞培養容器及びそれを利用した細胞培養方法
WO2007052653A1 (fr) * 2005-10-31 2007-05-10 Strex Incorporation Recipient de culture et appareil de culture
JP2008271912A (ja) * 2007-05-02 2008-11-13 Cellseed Inc 密閉系細胞培養容器及びそれを利用した細胞培養方法
JP2011519557A (ja) * 2008-05-05 2011-07-14 ウィルソン ウォルフ マニュファクチャリング コーポレイション 細胞容器
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Publication number Priority date Publication date Assignee Title
JP2020505030A (ja) * 2017-01-16 2020-02-20 レアクト４ライフ エッセエッレエッレ バイオリアクタおよびこのようなバイオリアクタの使用方法
JP7257043B2 (ja) 2017-01-16 2023-04-13 レアクト４ライフ エッセエッレエッレ バイオリアクタおよびこのようなバイオリアクタの使用方法
CN112300929A (zh) * 2019-07-31 2021-02-02 上海新微技术研发中心有限公司 一种微流控实验板及双面细胞培养方法

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