WO2019214465A1 - Dispositif jetable à membrane de culture cellulaire à lit fluidisé rempli de supports lamellés - Google Patents

Dispositif jetable à membrane de culture cellulaire à lit fluidisé rempli de supports lamellés Download PDF

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Publication number
WO2019214465A1
WO2019214465A1 PCT/CN2019/084600 CN2019084600W WO2019214465A1 WO 2019214465 A1 WO2019214465 A1 WO 2019214465A1 CN 2019084600 W CN2019084600 W CN 2019084600W WO 2019214465 A1 WO2019214465 A1 WO 2019214465A1
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WO
WIPO (PCT)
Prior art keywords
cell culture
closed bag
sheet
bag body
fluidized bed
Prior art date
Application number
PCT/CN2019/084600
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English (en)
Chinese (zh)
Inventor
甄宝贵
李驰
吴菲菲
李昌武
王树华
Original Assignee
武汉赛科成科技有限公司
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Publication date
Application filed by 武汉赛科成科技有限公司 filed Critical 武汉赛科成科技有限公司
Priority to JP2020600192U priority Critical patent/JP3233421U/ja
Publication of WO2019214465A1 publication Critical patent/WO2019214465A1/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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/14—Bags
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/28—Constructional details, e.g. recesses, hinges disposable or single use
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C12M25/20—Fluidized bed
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution

Definitions

  • the invention relates to the technical field of cell culture, in particular to a fluidized bed cell culture disposable membrane device filled with a sheet carrier.
  • the bioreactor currently undergoing cell culture is mainly divided into a fluidized bed bioreactor and a fixed bed bioreactor.
  • a fixed bed bioreactor is a cell adherent culture by loading a filler in the middle of a fixed container, the filler is composed of a cell culture carrier, and the cell culture carrier is usually a sheet fiber carrier having a diameter of 2 to 11 mm.
  • Cell-attached on a sheet-shaped carrier for fixed-bed culture was first established by NBS, USA, but its sheet-like carrier is usually placed in a bioreactor tank by wire mesh, and the cells are in a static culture for a long period of time.
  • the sheet-shaped carrier cannot be suspended, and after a long period of time, metabolites (hazardous substances) produced by the cells are aggregated in the sheet-like carrier and cannot be discharged, and it is difficult to achieve large-scale culture of the adherent cells, and it is difficult to directly perform cell sampling.
  • a fluidized bed reactor is a reactor that uses a gas or liquid to pass a granular solid layer to cause a solid particle to be in a suspended motion state, and performs a gas solid phase reaction process or a liquid solid phase reaction process.
  • the purpose of the fluidized bed is to make a fluidized bed Large bubble breakage, improve the contact of gas-solid phase, reduce back-mixing, improve the reaction effect, but when the cells are cultured, the cells are easily damaged by large bubbles, the ventilation distribution is uneven, and the mass transfer and oxygen transmission are poor, resulting in cells.
  • the growth state is not good.
  • the object of the present invention is to provide a fluidized bed cell culture disposable membrane device filled with a sheet carrier, which can remove metabolites in time, improve the cell culture environment on the sheet carrier, promote cell growth, and allow the adherent cells to obtain suspension. Cultivate a similar culture mechanism while maintaining the excellent specific effects of adherent cells and facilitating industrial amplification of adherent cells.
  • a fluidized bed cell culture disposable membrane device filled with a sheet carrier comprising a closed bag body, and the closed bag body is filled with a plurality of sheet carriers
  • An extended side seal is disposed on both sides of the outer side of the closed bag body, and each of the extended side seals is provided with a fixing mechanism, and the closed bag body is sealed by two sides
  • a fixing mechanism is fixed in the biological cell culture shaker, and an upper air inlet, an air outlet, a sampling port and at least one liquid inlet and the liquid inlet are provided on the top surface of the closed bag body, in the closed a bottom air inlet is provided on the bottom surface of the bag body, and a sterile quick connector is connected to the upper air inlet, the air outlet, the sampling port, the liquid inlet, the liquid outlet and the lower air inlet, in each of the A partitioning device is further disposed in the liquid outlet, and the closed bag body intercepts the sheet carrier in the closed bag through a partition device disposed at
  • the closed bag body is composed of an upper bag piece and a lower bag piece.
  • one or more sensor connectors are further disposed on the bottom surface of the closed bag body; preferably, two sensor connectors are disposed on the bottom surface of the closed bag body, one of which is for accessing the PH sensor probe The sensor connector and the other sensor connector for accessing the dissolved oxygen sensor probe.
  • the contact sensor connector includes a welding port, a buckle and a valve block
  • the welding port is welded to the surface of the closed bag body
  • the buckle is engaged with the welding port
  • the valve block The partition is disposed at a contact portion of the welding port and the closed bag body, wherein the buckle is used for fixing the sensor probe in the welding port, and the sensor probe is directly in contact with the culture liquid in the closed bag body through the block partition.
  • the fixing mechanism is a fiberglass rod enclosed in the extended side seal or a fixing hole formed on the extended side seal.
  • the separation device is a medical grade plastic separator.
  • the biological cell culture shaker is composed of a shaker control box, a tray, and a shaker seat that can be rocked up and down or shaken left and right, the shaker seat is fixed on the top of the shaker control box, and the tray is set at The top of the shaker seat is respectively fixed with a locking device on both sides of the bottom wall of the tray.
  • closed bag body, the extended side seal and the aseptic quick joint are all made of medical grade plastic material.
  • each of the sheet carriers comprises a plurality of sheet-like fiber blades which are arc-connected and made of polyester fiber and/or polypropylene material, and the plurality of sheets which are connected in an arc shape The fiber-like blades are flared outwardly.
  • the sheet carrier has a loading of 1 g/L to 100 g/L.
  • the advantages of the present invention are as follows: (1) by filling a sheet-shaped carrier in a closed bag, attaching the cells to the sheet-shaped carrier, and introducing the cell culture solution into the closed bag to make the tablet The carrier is suspended together with the cells adherent on the sheet carrier, and both the cell adherent culture and the suspension culture are used to obtain higher cell culture density, and the expanded culture of the adherent cells is also facilitated; (2) The ventilation method combining bottom ventilation and surface gas phase combination increases the traveling path of oxygen in the liquid and increases the dissolved oxygen amount; (3) the sheet carrier can act like a baffle and can pass The bubbles generated by the gas into the liquid are cut off, effectively preventing the bubbles from directly rupturing in the liquid, the risk of injuring the cells, and reducing the damage to the cells; (4) filling the sheet carrier by filling the closed bag body After the sheet-shaped carrier, the oxygen transfer and mass transfer path of the cell culture liquid is increased, so that the nutrients in the liquid can be better and more fully utilized by the cells
  • Figure 1 is a top plan view of a bag body according to a first embodiment of the present invention
  • FIG. 2 is a schematic bottom view of a bag body according to a second embodiment of the present invention.
  • FIG. 4 is a perspective view of a bag body and a shaker according to a first embodiment of the present invention
  • Figure 7 is a perspective view of a bag body according to a second embodiment of the present invention.
  • Figure 8 is a perspective view of a bag body and a shaker according to a second embodiment of the present invention.
  • Figure 9 is a schematic view showing the connection pipe of the top surface of the bag body in the state of use according to the first embodiment of the present invention.
  • Figure 12 is a schematic view showing the structure of a sheet carrier in the present invention.
  • a fluidized bed cell culture disposable membrane device filled with a sheet carrier comprises a closed bag body 1 filled with a plurality of pieces inside the closed bag body 1 .
  • the carrier 2 is provided with an extended side seal 3 on both sides of the outer side of the closed bag body 1, and a fixing mechanism is arranged on each of the extended side seals 3, and the closed bag body 1 is extended by the side edges.
  • the fixing mechanism provided is fixed in the biological cell culture shaker 15, and an upper air inlet 6, an air outlet 7, a sampling port 8, a liquid inlet 9 and a top are arranged on the top surface of the closed bag body 1.
  • the liquid inlet port 10 is provided with a lower air inlet 11 on the bottom surface of the closed bag body 1, and the upper air inlet port 6, the air outlet port 7, the sampling port 8, the liquid inlet port 9, the liquid outlet port 10 and the lower air inlet.
  • the mouth 11 is connected with a sterile quick joint 12, and a partitioning device 13 is further disposed in the liquid outlet 10; the closed bag body 1 encloses the sheet carrier 2 in a closed state by a partitioning device 13 disposed in the liquid outlet 10
  • the separation of the digested cells and the sheet carrier 2 is achieved, that is, the cells are detached from the sheet carrier 2 during digestion, and the cells are discharged.
  • the sheet carrier 2 When the mouth 10 is output, the sheet carrier 2 is trapped in the closed bag 1 through the partitioning means 13 in the liquid outlet 10, and the cells are discharged from the liquid outlet 10 together with the cell culture liquid 16 to a larger organism. In the cell culture tank, industrialized amplification production of adherent cells is realized.
  • the partitioning device 13 is a medical grade plastic separating net, and the mesh size of the dividing net is such that the cells can be allowed to pass, and the sheet carrier 2 is not allowed to pass.
  • one or more sensor connectors 14 may be provided on the bottom surface of the closed bag body 1 according to the actual cell culture environment requirements, or the sensor connector 14 may not be provided, for example, When the disposable membrane device provided by the invention is placed in a constant carbon dioxide incubator for small-scale cell culture, the sensor connector 14 can be omitted.
  • the sensor connector 14 When it is required to provide the sensor connector 14 on the bottom surface of the closed bag body 1, it is preferable to provide two sensor connectors as shown in FIG. 2 or FIG. 6, one of which is a sensor connector for accessing the pH sensor probe. 141, the other is a sensor connector 142 for accessing the oxygen sensor probe.
  • the sensor connector 14 is a contact sensor connector.
  • the contact sensor connector includes a soldering port 1401, a buckle 1402, and a vane partition 1403.
  • the soldering port 1401 is directly soldered to the surface of the closed bag body 1.
  • the corresponding sensor probe is fixedly disposed in the buckle 1402, and one end extends into the welding port 1401 and passes through the valve partition 1403 to directly contact the cell culture liquid in the closed bag body 1 and the other end passes through the buckle 1402. It is engaged with the welding port 1401.
  • the biological cell culture shaker 15 is composed of a shaker control box 151, a tray 153, and a shaker seat 152 which can be rocked up and down or left and right, and the shaker seat 152 is fixed to the shaker control.
  • the tray 153 is disposed at the top of the rocker seat 152, and a locking device 154 is respectively fixed on both sides of the bottom wall of the tray 151.
  • the locking device 154 is according to actual needs.
  • the closed bag body 1 is fixed by the locking device 154 provided on both sides of the inner bottom wall of the tray 153.
  • the shaker seat 152 is controlled to swing up and down or left and right by the shaker control box 151 to drive the tray 153 to shake with the closed bag 1 fixed on the tray 153, so that the cell culture solution 13 is
  • the sheet carrier 2 is suspended in the closed bag body 1 to sufficiently contact the cell culture solution 13 with the cells attached to the sheet carrier 2 and to reduce the accumulation of metabolites (hazardous substances) produced by the cells on the sheet carrier 2. , thereby improving the sheet carrier 2 Amplifying cell culture environment, promote the growth of cells, adherent cells so obtained suspension culture cultured similar mechanism, while maintaining excellent effects related adherent cells, adherent cells facilitates industry.
  • each of the sheet-like carriers 2 comprises a plurality of sheet-like fiber blades 2a which are arc-connected and made of polyester fiber and/or polypropylene material, and some of which are curved.
  • the connected sheet-like fiber blades 2a are arranged to be flared outward.
  • the loading of the sheet-like carrier 2 in the closed bag body 1 can be set according to the capacity requirement of the closed bag body 1, and the loading range thereof is specifically 1 g/L-100 g/L.
  • a plurality of liquid inlets 9 and a plurality of liquid outlets 10 may be disposed on the top and bottom surfaces of the closed bag body 1, as shown in FIG.
  • the upper air inlet 6 can be quickly connected to the external air intake pipeline through the aseptic quick connector 12 as shown in FIG. 9, and the external air intake pipeline can sequentially include a medical silicone tube 18, an air filter 20, and then connect the external air.
  • Supply device (not shown);
  • the air outlet 7 can be quickly connected to the external exhaust line via the aseptic quick connector 12 as shown in FIG. 9.
  • the external exhaust line can sequentially include a medical silicone tube 18, an air filter 20, and then connected to the outside atmosphere or gas collection. Device (not shown);
  • the liquid outlet 10 can be quickly butted to the external liquid outlet pipe through the aseptic quick connector 12 as shown in FIG. 9, and the external liquid inlet pipe can include the medical heat shrinkable tube 22, the blind plug 23, and then the external liquid collecting device. (not shown in the figure);
  • the lower air inlet 11 can be quickly butted to the external air intake pipe via the aseptic quick connector 12 as shown in FIG. 10, and the external air intake pipe can sequentially include a medical silicone tube 18, an air filter 20, and then connected to an external gas supply device ( Not shown in the figure).
  • the medical silicone tube 18 and the medical heat-shrinkable tube 22 on each of the tubes are provided with a Y-shaped clip 19 for controlling the ingress and egress of liquid and gas, and the respective components of the tube are sealedly connected by a cable tie 17.

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Abstract

La présente invention concerne un dispositif jetable à membrane de culture cellulaire à lit fluidisé rempli de supports lamellés, comprenant un sac fermé. L'intérieur du sac fermé est rempli de plusieurs supports lamellés; chaque côté de l'extérieur du sac fermé est pourvu d'un joint latéral étendu; le sac fermé est fixé dans un agitateur de culture cellulaire biologique au moyen de mécanismes de fixation disposés sur les joints latéraux étendus sur les deux côtés; la surface supérieure du sac fermé est pourvue d'une entrée d'air supérieure, d'une sortie d'air, d'un orifice d'échantillonnage et d'au moins une entrée de liquide et d'au moins une sortie de liquide; la surface inférieure du sac fermé est pourvue d'une entrée d'air inférieure; l'entrée d'air supérieure, la sortie d'air, l'orifice d'échantillonnage, l'entrée de liquide, la sortie de liquide et l'entrée d'air inférieure sont chacune reliées à un raccord rapide stérile; un dispositif de séparation est en outre disposé dans chaque sortie de liquide; le sac fermé retient les supports lamellés dans le sac fermé au moyen du dispositif de séparation disposé au niveau de la sortie de liquide de façon à séparer les cellules des supports lamellés. La présente invention présente des avantages tels qu'un bon effet de dissolution d'oxygène, une densité finale de culture cellulaire importante et une praticité pour des cellules adhérentes à grande échelle industrielle.
PCT/CN2019/084600 2018-05-09 2019-04-26 Dispositif jetable à membrane de culture cellulaire à lit fluidisé rempli de supports lamellés WO2019214465A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2020600192U JP3233421U (ja) 2018-05-09 2019-04-26 シート状担体を充填するための流動床細胞培養使い捨て膜装置

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CN201810436720.0 2018-05-09
CN201810436720.0A CN108315259B (zh) 2018-05-09 2018-05-09 一种填充片状载体的流化床细胞培养一次性膜装置

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CN111500453A (zh) * 2020-04-30 2020-08-07 龙岩学院 一种生物细胞培育用密封培育装置及其使用方法
WO2021114087A1 (fr) * 2019-12-10 2021-06-17 苏州生动细胞生物科技有限公司 Poche de culture cellulaire de faible volume
CN114317397A (zh) * 2021-12-02 2022-04-12 罗火生 一种纸片载体固定床培养细胞传代罐传罐放大方法

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CN108315259B (zh) * 2018-05-09 2024-01-23 武汉赛科成科技有限公司 一种填充片状载体的流化床细胞培养一次性膜装置
CN108998344B (zh) * 2018-08-06 2022-07-05 武汉赛科成科技有限公司 一种连续大规模生产重组腺病毒的方法
CN112899165A (zh) * 2019-11-19 2021-06-04 上海三维生物技术有限公司 一种高密度贴壁细胞培养体系的细胞接种方式
WO2021114085A1 (fr) * 2019-12-10 2021-06-17 苏州生动细胞生物科技有限公司 Ensemble de culture cellulaire pouvant être fixé

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EP2379699B1 (fr) * 2008-12-19 2017-12-13 Stobbe Tech A/S Dispositif pour la culture industrielle sur biocouches
CN201933088U (zh) * 2011-01-14 2011-08-17 南京元创生物工程有限公司 片状载体固流床培养装置
CN205420436U (zh) * 2015-12-23 2016-08-03 上海吉凯基因科技有限公司 一种细胞培养袋
CN105925481A (zh) * 2016-05-16 2016-09-07 上海吉凯基因科技有限公司 全自动细胞连续培养系统
CN108315259A (zh) * 2018-05-09 2018-07-24 武汉赛科成科技有限公司 一种填充片状载体的流化床细胞培养一次性膜装置
CN208454993U (zh) * 2018-05-09 2019-02-01 武汉赛科成科技有限公司 一种填充片状载体的流化床细胞培养一次性膜装置

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Publication number Priority date Publication date Assignee Title
WO2021114087A1 (fr) * 2019-12-10 2021-06-17 苏州生动细胞生物科技有限公司 Poche de culture cellulaire de faible volume
CN111500453A (zh) * 2020-04-30 2020-08-07 龙岩学院 一种生物细胞培育用密封培育装置及其使用方法
CN114317397A (zh) * 2021-12-02 2022-04-12 罗火生 一种纸片载体固定床培养细胞传代罐传罐放大方法

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