WO2019245050A1 - Dispositif de culture de cellules à fibres creuses, procédé de culture de cellules et procédé de production de surnageant de culture - Google Patents

Dispositif de culture de cellules à fibres creuses, procédé de culture de cellules et procédé de production de surnageant de culture Download PDF

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Publication number
WO2019245050A1
WO2019245050A1 PCT/JP2019/024872 JP2019024872W WO2019245050A1 WO 2019245050 A1 WO2019245050 A1 WO 2019245050A1 JP 2019024872 W JP2019024872 W JP 2019024872W WO 2019245050 A1 WO2019245050 A1 WO 2019245050A1
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WIPO (PCT)
Prior art keywords
hollow fiber
culture
culture solution
cell
filter housing
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PCT/JP2019/024872
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English (en)
Japanese (ja)
Inventor
漆畑　直樹
勝幸 隠岐
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株式会社バイオミメティクスシンパシーズ
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Application filed by 株式会社バイオミメティクスシンパシーズ filed Critical 株式会社バイオミメティクスシンパシーズ
Priority to CN201980006288.4A priority Critical patent/CN111448305B/zh
Publication of WO2019245050A1 publication Critical patent/WO2019245050A1/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
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means

Definitions

  • the solution described in the present specification is capable of culturing cells stably by culturing the cells inside the hollow fiber and circulating the culture solution outside the hollow fiber. Based on the knowledge that high-quality cells and culture supernatants can be obtained inexpensively and in large quantities.
  • This device further has a cell introduction part 5,
  • the cell introduction part 5 is a cylindrical volume part having a space volume provided at a part corresponding to the hollow fiber filter connecting the first end cap 6a, and one end of the cylindrical volume part has: A pusher with a gasket is inserted, and the other end of the cylindrical volume is connected to a hollow fiber filter. By pushing the pusher in the direction of the hollow fiber filter, cells are inserted into the lumen of the hollow fiber filter.
  • the device is introduced.
  • This manufacturing method A cell introduction step of introducing cells into the hollow fiber 3 via the cell introduction part 5; A culture solution supply step of supplying a culture solution into the filter housing via the culture solution introduction unit 7; A culture solution discharging step of discharging the culture solution inside the filter housing from the culture solution discharge section 9, and a step of culturing cells inside the hollow fiber 3; A culture supernatant collecting step of collecting a culture supernatant from the discharged liquid obtained in the culture liquid discharging step; And a method for producing a culture supernatant.
  • FIG. 1 is a conceptual diagram showing a configuration example of a culture device.
  • the apparatus comprises: a filter housing 1, a hollow fiber 3 existing inside the filter housing, a first end cap 6 a, a culture solution introduction unit 7, and a culture solution discharge unit 9. Having.
  • Tissues from which mesenchymal stem cells are separated include umbilical cord, cord blood, bone marrow, amniotic membrane, placenta, dental pulp, fat, cartilage, menstrual blood, breast milk, etc., and the source tissues and animal species are particularly limited Not something.
  • mesenchymal stem cells must have been transduced or modified to establish immortalized cell lines, to provide cell characteristics that can withstand long-term culture, or to improve the effectiveness of culture supernatants. May be used.
  • the cellulosic water-soluble polymer methylcellulose (MC) or carboxymethylcellulose (CMC) to the culture medium.
  • Other cellulose derivatives, or other water-soluble polymers such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), or sodium alginate and their gelling agents, using a technique to increase the viscosity of the culture solution. May be.
  • the concentration of the water-soluble polymer can be set with reference to the previous report. As a result, it is possible to form spheroids of uniform size in the cell culture space in the hollow fiber and to arrange them uniformly in space.
  • the cell introduction section 5 is at least composed of an outer cylinder, a gasket, a pusher, and a lock mechanism.
  • the cell introduction section 5 is at least composed of an outer cylinder, a gasket, a pusher, and a lock mechanism.
  • connection mode of the device can be selected according to the purpose of the practitioner. For example, when connecting tubes and the like that form the culture medium flow path, when using existing aseptic connection technology often used in the bioindustry, or when incorporating a stainless steel container as a culture medium storage container, such a connection is required.
  • the connection mode of the device according to the above can be taken.
  • this technology is used in the production of general biotechnology-based drugs, etc. The technology may be designed by adopters.
  • a cell-based water-soluble polymer such as methylcellulose MC and carboxymethylcellulose CMC, other cellulose derivatives, or other water-soluble polymers such as polyethylene glycol PEG and polyvinylpyrrolidone are used to suppress cell aggregate formation.
  • a method of increasing the viscosity of the culture solution by adding PVP or sodium alginate or a gelling agent thereof may be used.
  • the cell introduction part 5 is removed, and the inside of the hollow fiber 3 and the inside of the filter housing 1 are hermetically sealed by attaching an end cap 6a to the end 1a of the filter housing and the end 4a of the hollow fiber if there is a gasket or a sealing material.
  • This is designated as component 2.
  • the hollow fiber filter module should be kept horizontal to avoid bias of cells inside the hollow fiber.
  • the entire apparatus of the present invention may be installed in a chamber of a constant temperature apparatus, or may be controlled by a jacket type heating apparatus for heating.
  • heating is performed in the vicinity of the culture solution supply path 8 and the culture solution introduction unit 7 on the side where the culture medium is sent to the hollow fiber filter module, and the temperature is adjusted to the optimum temperature immediately before the culture solution is sent to the hollow fiber filter module.
  • the hollow fiber filter module also has a mechanism for maintaining the optimum temperature, and further includes a culture medium discharge section 9 and a culture medium discharge path 10 on the side where the culture medium is discharged from the hollow fiber filter module. May be cooled to 4 to 10 ° C., and the culture solution storage container may be cooled to 4 to 10 ° C. to prevent denaturation and degradation of the medium components and cell secretion components in long-term culture.
  • a substantially feasible method for recovering a culture supernatant containing secretions of cells is as follows.
  • a culture solution on the side where cells are present is circulated through a hollow fiber membrane to collect the culture solution on the same side as a culture supernatant (Patent Document 1).
  • the culture supernatant is collected from the outside of the hollow fiber by diffusion from the hollow fiber lumen through which the cells are closed and there is no active circulation to the outside of the hollow fiber through the hollow fiber membrane.
  • the culture supernatant may be obtained by continuous culture such as fed-batch culture or perfusion culture.
  • the filter housing ends 1a and 1b have a sanitary connection port shape, and a hollow fiber filter module (PES MidiKros TC module / 0.2 ⁇ m fractionation size, filter membrane area) with a Luer lock connect at the culture solution introduction and culture solution discharge portions. 290 cm 2 , total length 44 cm; T04-P20U-05-N; Spectrum Laboratories) was used.
  • An end cap 6b having a ferrule sanitary connection port and including a silicon gasket and a silicone sealant was closed tightly by clamping so as to close the filter housing end 1b and the hollow fiber end 4b.
  • the collected culture supernatant is filtered through a 0.2 ⁇ m PES syringe filter (25 mm GD / X syringe filter (PES 0.2 ⁇ m sterilized); 6896-2502; GE Healthcare Japan) and used for analysis. Stored frozen at -28 ° C.
  • the culture medium was completely exchanged once every 3 to 4 days, and the culture medium was exchanged at 7 days after the start of culture (Day 7), 11 days after the start of culture (Day 11), and 14 days after the start of culture.
  • the culture supernatant was collected before replacing the medium.
  • These culture supernatants were filtered through a 0.2 ⁇ m PES syringe filter (25 mm GD / X syringe filter (PES 0.2 ⁇ m sterilized); 6896-2502; GE Healthcare Japan) until they were used for analysis. Stored frozen at °C.
  • the concentration of HGF protein which was another index, decreased at the upper limit of 5,794 pg / mL on Day 7, suggesting that the quality of the recovered culture supernatant deteriorated in long-term culture exceeding 1 week.
  • the maximum secreted amount on Day 35 reaches 7,391 pg / mL, and 5,000 The period of recovery at pg / mL or higher was very long, from Day 21 to Day 50.
  • Glucose the main energy source of the cells, was quantified to evaluate changes in the concentration of the medium during long-term culture.
  • a commercially available kit was used for glucose determination according to the manual (Glucose Colorimetric Assay Kit 2; BioVision). The results are shown in Table 4 and FIG.

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

Abstract

Le problème décrit par la présente invention est de fournir un dispositif de culture. La solution selon l'invention porte sur un dispositif de culture de cellules à fibres creuses pour produire un surnageant de culture, ledit dispositif de culture comprenant : un boîtier de filtration (1); des fibres creuses (3) présentes dans le boîtier de filtration et ayant une taille de pore de 5 nm à 1 µm inclus, dans lesquelles des cellules sont introduites par l'intermédiaire d'une partie d'introduction (5); un premier capuchon d'extrémité (6a) pour fermer hermétiquement une première extrémité (4a) des fibres creuses (3) et du boîtier de filtration (1); une partie d'introduction (7) de liquide de culture pour introduire un liquide de culture dans le boîtier de filtration; et une partie d'évacuation (9) de liquide de culture pour évacuer le liquide de culture ayant traversé l'intérieur du boîtier de filtration.
PCT/JP2019/024872 2018-06-22 2019-06-24 Dispositif de culture de cellules à fibres creuses, procédé de culture de cellules et procédé de production de surnageant de culture WO2019245050A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201980006288.4A CN111448305B (zh) 2018-06-22 2019-06-24 中空纤维细胞培养装置、细胞培养方法、培养上清液的制造方法

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JP2018-119267 2018-06-22
JP2018119267A JP6469287B1 (ja) 2018-06-22 2018-06-22 中空糸細胞培養装置，細胞培養方法，培養上清の製造方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021200744A1 (fr) * 2020-03-31 2021-10-07 Ｃｅｌｌ Ｅｘｏｓｏｍｅ Ｔｈｅｒａｐｅｕｔｉｃｓ株式会社 Procédé de production de cellules prolifératives, procédé de production d'un produit cellulaire, population de cellules souches mésenchymateuses et son procédé de production, surnageant de culture de cellules souches et son procédé de production, et agent thérapeutique

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11911553B2 (en) * 2018-07-06 2024-02-27 Fenwal, Inc. Systems and methods for concentrating cells with a syringe and a centrifuge
TWI780616B (zh) * 2021-03-04 2022-10-11 國立清華大學 灌流式細胞培養裝置及灌流式細胞培養系統
WO2023190448A1 (fr) * 2022-03-29 2023-10-05 国立大学法人東海国立大学機構 Procédé de culture de cellules souches mésenchymateuses

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005333945A (ja) * 2004-05-31 2005-12-08 Toray Ind Inc 培養用中空糸モジュール及び培養装置及び細胞製剤の製造方法
WO2016148230A1 (fr) * 2015-03-17 2016-09-22 東洋紡株式会社 Méthode pour produire un surnageant de culture de cellules souches
WO2017104558A1 (fr) * 2015-12-15 2017-06-22 東洋紡株式会社 Membrane à fibres creuses et module à fibres creuses pour culture cellulaire

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986002379A1 (fr) * 1984-10-09 1986-04-24 Endotronics, Inc. Dispositif de culture a fibres creuses pour ameliorer la perfusion de substances nutritives et la concentration des produits, et procede de fonctionnement
CN1118563C (zh) * 2000-12-08 2003-08-20 中国医学科学院血液学研究所 中空纤维模拟骨髓造血装置
JP2004283010A (ja) * 2003-03-19 2004-10-14 Toyobo Co Ltd 細胞の培養方法、細胞培養物及び医用生体材料
JP3726188B2 (ja) * 2002-05-28 2005-12-14 東洋紡績株式会社 中空糸付き器具及びその使用方法
JP2007202430A (ja) * 2006-01-31 2007-08-16 Scimedia Ltd 細胞培養用中空糸束モジュール
JP5496901B2 (ja) * 2008-09-26 2014-05-21 旭化成ケミカルズ株式会社 清澄化されたバイオ医薬培養液を製造するための多孔質中空糸膜の使用
JP2014060991A (ja) * 2012-08-30 2014-04-10 Asahi Kasei Corp 多孔質中空糸の内腔を用いる幹細胞の培養方法
JP2017158488A (ja) * 2016-03-10 2017-09-14 東洋紡株式会社 細胞回収方法
JP2018014947A (ja) * 2016-07-29 2018-02-01 東洋紡株式会社 中空糸膜モジュールを用いる細胞培養方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005333945A (ja) * 2004-05-31 2005-12-08 Toray Ind Inc 培養用中空糸モジュール及び培養装置及び細胞製剤の製造方法
WO2016148230A1 (fr) * 2015-03-17 2016-09-22 東洋紡株式会社 Méthode pour produire un surnageant de culture de cellules souches
WO2017104558A1 (fr) * 2015-12-15 2017-06-22 東洋紡株式会社 Membrane à fibres creuses et module à fibres creuses pour culture cellulaire

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021200744A1 (fr) * 2020-03-31 2021-10-07 Ｃｅｌｌ Ｅｘｏｓｏｍｅ Ｔｈｅｒａｐｅｕｔｉｃｓ株式会社 Procédé de production de cellules prolifératives, procédé de production d'un produit cellulaire, population de cellules souches mésenchymateuses et son procédé de production, surnageant de culture de cellules souches et son procédé de production, et agent thérapeutique
EP4155389A3 (fr) * 2020-03-31 2023-07-12 Cell Exosome Therapeutics Inc. Procédé de production de cellules proliférantes, méthode de production de produit cellulaire, population de cellules souches mésenchymateuses et méthode de production

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CN111448305B (zh) 2023-09-29
JP2019216694A (ja) 2019-12-26
JP6469287B1 (ja) 2019-02-13
CN111448305A (zh) 2020-07-24

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