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WO2025164695A1 - Dispositif de culture cellulaire tridimensionnel et procédé de culture cellulaire l'utilisant - Google Patents

Dispositif de culture cellulaire tridimensionnel et procédé de culture cellulaire l'utilisant

Info

Publication number
WO2025164695A1
WO2025164695A1 PCT/JP2025/002887 JP2025002887W WO2025164695A1 WO 2025164695 A1 WO2025164695 A1 WO 2025164695A1 JP 2025002887 W JP2025002887 W JP 2025002887W WO 2025164695 A1 WO2025164695 A1 WO 2025164695A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell culture
pump
cell
cells
culture device
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
PCT/JP2025/002887
Other languages
English (en)
Japanese (ja)
Inventor
亜紀 蒔苗
孝尚 鈴木
欣也 栗原
和希 中本
充 屋代
勉 岩本
伸之 川島
明日菜 杉本
佳代子 大西
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.)
NEPA GENE CO Ltd
Institute of Science Tokyo
Original Assignee
NEPA GENE CO Ltd
Institute of Science Tokyo
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
Application filed by NEPA GENE CO Ltd, Institute of Science Tokyo filed Critical NEPA GENE CO Ltd
Publication of WO2025164695A1 publication Critical patent/WO2025164695A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a cell culture device and a cell culture method using the same.
  • the object of the present invention is to provide a cell culture device capable of applying one or more physical and/or chemical stimuli to cells being cultured, and to provide cell culture cells using the same.
  • a cell culture device comprising a cell culture area, a first pump connected to the cell culture area, and a second pump connected to the cell culture area, wherein the first pump, the cell culture area, and the second pump form a line connected in this order, and the first pump and the second pump can move fluid in forward and reverse directions, respectively, independently of each other, and the pressures at which the fluids are moved can be varied independently of each other.
  • the cell culture device according to (1) further comprising a liquid culture medium reservoir container incorporated in the line, wherein the liquid culture medium contained in the liquid culture medium reservoir container is supplied to the cell culture area by the first pump and/or the second pump.
  • the cell culture device according to (1) or (2), further comprising a cell culture component container arranged upstream of the first pump, wherein one or more cell culture components contained in the cell culture component container are supplied to the liquid culture medium contained in the liquid culture medium reserve container by the first pump and/or the second pump.
  • the cell culture device according to (3), wherein the one or more cell culture components are supplied in the form of a gas to the liquid culture medium contained in the liquid culture medium reservoir container.
  • a cell culture device according to any one of (1) to (5), further comprising a cell collection column incorporated in the line, wherein the first pump and/or the second pump detach the cultured cells on the cell culture area, and the detached cultured cells are collected in the cell collection column.
  • the cell culture device further comprising a cell collection container connected to the cell collection column, wherein cultured cells separated from the cell collection column are collected into the cell collection container by the first pump and/or the second pump.
  • the cell culture device according to any one of (1) to (7), wherein the cell culture area is coated with a polymer that makes it easy for cells cultured on the cell culture area to detach from the cell culture area at low temperatures, and the cells are cultured on the polymer.
  • the cell culture device according to any one of (1) to (8), further comprising an observation camera, a pH meter, a dissolved oxygen meter, a flow meter, and a weight sensor incorporated in the line.
  • a method for culturing cells comprising culturing cells on the cell culture region of the cell culture device according to any one of (1) to (9).
  • FIG. 1 is a schematic diagram showing a preferred embodiment of the cell culture device of the present invention.
  • FIG. 1 is a diagram schematically illustrating an example in which AI and IoT are applied to the culture method of the present invention.
  • the cell culture device shown in Figure 1 includes a cell culture region 10.
  • a first pump 12 is connected upstream of the cell culture region 10.
  • a second pump 14 is connected downstream of the cell culture region 10.
  • the first pump 12, cell culture region 10, and second pump 14 form a line connected in this order.
  • the first pump 12 and second pump 14 can move fluid in the forward and reverse directions, respectively, independently of each other, and the pressures at which the fluids are moved can be varied independently of each other.
  • a liquid culture medium reservoir container 16 for storing culture medium is installed in the line between the first pump 12 and the cell culture area 10.
  • the specific example shown in FIG. 1 further includes a cell culture component container located upstream of the first pump 12.
  • three cell culture component containers 18a, 18b, and 18c are included.
  • the three cell culture component containers 18a, 18b, and 18c contain, for example, a stimulant solution, a buffer solution (PBS), and a liquid culture medium, respectively.
  • PBS buffer solution
  • the three cell culture component containers 18a, 18b, and 18c are preferably stored in a refrigerator 22.
  • a cell container 24 is placed upstream of the cell culture area 10 via a three-way cock 23 and a solenoid valve 21.
  • a cell recovery column 26 is further incorporated into the line between the cell culture region 10 and the second pump 14.
  • a cell collection container 40 is connected upstream of the cell collection column 26 via a three-way cock 36 and a solenoid valve 38.
  • the solenoid valve 38 is connected to the cell culture region 10 via a three-way cock 42 and a solenoid valve 44 downstream of the cell culture region 10.
  • two pH meters 28, 29, two dissolved oxygen meters 30, 31, two flow meters 32, 34, and two pressure sensors 46, 48 are incorporated into pressure chambers 47, 50 in the line.
  • the pressure chambers 47, 50 have a damping effect caused by the expansion and compression of gas within the chamber, which serves to suppress pulsation of fluids such as liquid culture medium pumped from the first pump 12.
  • the cell culture area 10 may be a flat surface covered with a solid medium such as agar or agarose gel, but it is preferable that the surface of the flat surface is coated with a polymer that facilitates detachment from the cell culture area at low temperatures.
  • a polymer that facilitates detachment from the cell culture area at low temperatures.
  • Such polymers are well known and are described, for example, in Patent Document 2.
  • the polymer described in Patent Document 2 When the polymer described in Patent Document 2 is coated on a flat surface, the polymer becomes hydrophilic at temperatures below 20°C, making it easier for cells to detach naturally, and becomes hydrophobic at temperatures above 25°C, allowing cells to adhere and proliferate.
  • the first pump 12 and second pump 14 are operated in sequence (flowing liquid from upstream to downstream) to send culture medium, stimulants, etc. from the cell culture component containers 18a, 18b, and 18c to the cell culture area 10 via the liquid culture medium reserve container 16.
  • the solenoid valve 21 is set so that liquid flows from the liquid culture medium reserve container 16 to the cell culture area 10.
  • a weight sensor 51 is provided below the cell culture component containers 18a, 18b, and 18c, and it is preferable that it has a function to measure weight and issue a warning when it is time to replace consumables.
  • During cell culture it is preferable to measure the pH and dissolved oxygen of the culture medium using pH meters 28 and 29 and the dissolved oxygen meters 30 and 31, and to culture the cells while appropriately adjusting the pH and dissolved oxygen concentration of the culture medium. Furthermore, during this process, cells can be cultured while measuring the flow rate of the liquid medium using flow meters 32, 34, and the pressure from first pump 12 and second pump 14 can be adjusted appropriately to apply shear stress to the cultured cells. It is also preferable to provide an observation camera 53 above and/or below the cell culture area to determine the differentiation state of the cultured cells, predict cell density and cell proliferation, and measure the presence and size of air bubbles.
  • cell culture area 10 When recovering cells, for example, cold phosphate buffered saline (PBS) stored in refrigerator 22 is pumped into cell culture area 10. At this time, solenoid valves 21 and 44 are switched so that the flow path connects to cell collection column 26. As described above, cell culture area 10 is coated with a polymer that causes cells to detach at temperatures below 20°C, and the detached cells are accumulated in cell collection column 26. If detachment is difficult, the first pump 12 and second pump 14 are repeatedly operated in forward and reverse directions to promote cell detachment by the water flow.
  • PBS cold phosphate buffered saline
  • the cells accumulated in the cell collection column 26 are sent to the cell collection container 40 by reversely pumping the cell collection liquid from the second pump 14. At this time, the electromagnetic valve 38 is set so that the liquid flows from the cell collection column 26 to the cell collection container 40.
  • the cell culture device of the present invention described above, by varying the liquid delivery volume (liquid delivery pressure) of the first pump 12 and the second pump 14, it is possible to apply physical stimuli such as shear stress to the cultured cells. Furthermore, by adjusting the desired gas concentration and bubbling it into the cell culture component containers 18b and 18c, it is possible to include one or more medium components, such as gas stimuli or stimulant drugs, in the culture medium. This allows multiple chemical and/or physical stimuli to be applied to the cultured cells simultaneously, making it possible to more closely resemble the in vivo environment. Furthermore, it is possible to automate the process from cell seeding to cell recovery after culture.
  • Data integration and analysis It integrates data on pressure, shear stress, pH, oxygen concentration, cell cycle, cell proliferation, cell differentiation, etc., and analyzes associations and correlations. Statistical methods and machine learning algorithms are used to extract patterns and trends from large amounts of data. This allows the status of the cell culture process to be monitored and stored in the cloud.
  • Pattern prediction and optimization It learns from accumulated data and builds predictive models for the cell culture process, enabling prediction and automatic control to optimize parameters such as pressure, shear stress, chemicals, gas stimulation, pH, oxygen concentration, cell cycle, cell proliferation, and cell differentiation.
  • the correlation coefficient can be used as a trigger to further optimize the culture conditions.
  • Real-time monitoring and remote control Real-time monitoring of cell culture processes and remote access to data and control commands allows for cell culture processes to be monitored from a remote location and controlled or adjusted as needed.
  • Real-time alerts and remote monitoring The system can monitor the status of cell culture from a remote location, detect abnormalities in the cell culture process and important events from various sensor information, and provide early warning of problems through real-time alert notifications, allowing appropriate measures to be taken as necessary.
  • FIG. 2 shows a schematic summary of the above-mentioned ways in which AI and IoT can be utilized.

Landscapes

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

Abstract

Sont divulgués un dispositif de culture cellulaire permettant d'appliquer un ou plusieurs stimuli physiques et/ou chimiques à des cellules pendant la culture, ainsi qu'un procédé de culture cellulaire l'utilisant. Ce dispositif de culture cellulaire est équipé d'une région de culture cellulaire, d'une première pompe reliée à la région de culture cellulaire et d'une seconde pompe reliée à la région de culture cellulaire. La première pompe, la région de culture cellulaire et la seconde pompe forment une ligne reliée dans cet ordre. La première pompe et la seconde pompe peuvent chacune déplacer un fluide dans la direction avant et la direction inverse indépendamment l'une de l'autre et les pressions auxquelles le fluide est déplacé sont variables et indépendantes l'une de l'autre.
PCT/JP2025/002887 2024-02-01 2025-01-30 Dispositif de culture cellulaire tridimensionnel et procédé de culture cellulaire l'utilisant Pending WO2025164695A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2024-013960 2024-02-01
JP2024013960 2024-02-01

Publications (1)

Publication Number Publication Date
WO2025164695A1 true WO2025164695A1 (fr) 2025-08-07

Family

ID=96590297

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2025/002887 Pending WO2025164695A1 (fr) 2024-02-01 2025-01-30 Dispositif de culture cellulaire tridimensionnel et procédé de culture cellulaire l'utilisant

Country Status (1)

Country Link
WO (1) WO2025164695A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11349643A (ja) * 1998-06-05 1999-12-21 Nitta Gelatin Inc 感温性高分子化合物およびその製造方法、感温性高分子組成物、細胞培養基材
JP2006314250A (ja) * 2005-05-12 2006-11-24 Hitachi Medical Corp 自動培養装置
JP2016530893A (ja) * 2013-09-16 2016-10-06 ジェンザイム・コーポレーション 細胞培養物を処理する方法およびシステム
JP2018110592A (ja) * 2014-07-22 2018-07-19 株式会社日立ハイテクノロジーズ 細胞分散装置およびそれを用いた自動継代培養システム
WO2022091648A1 (fr) * 2020-10-29 2022-05-05 株式会社アステック Dispositif de culture cellulaire et procédé de culture cellulaire

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11349643A (ja) * 1998-06-05 1999-12-21 Nitta Gelatin Inc 感温性高分子化合物およびその製造方法、感温性高分子組成物、細胞培養基材
JP2006314250A (ja) * 2005-05-12 2006-11-24 Hitachi Medical Corp 自動培養装置
JP2016530893A (ja) * 2013-09-16 2016-10-06 ジェンザイム・コーポレーション 細胞培養物を処理する方法およびシステム
JP2018110592A (ja) * 2014-07-22 2018-07-19 株式会社日立ハイテクノロジーズ 細胞分散装置およびそれを用いた自動継代培養システム
WO2022091648A1 (fr) * 2020-10-29 2022-05-05 株式会社アステック Dispositif de culture cellulaire et procédé de culture cellulaire

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