WO2022252003A1 - 密闭培养系统 - Google Patents

密闭培养系统 Download PDF

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Publication number
WO2022252003A1
WO2022252003A1 PCT/CN2021/097191 CN2021097191W WO2022252003A1 WO 2022252003 A1 WO2022252003 A1 WO 2022252003A1 CN 2021097191 W CN2021097191 W CN 2021097191W WO 2022252003 A1 WO2022252003 A1 WO 2022252003A1
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WIPO (PCT)
Prior art keywords
culture
closed
pipeline
room
bottle
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PCT/CN2021/097191
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English (en)
French (fr)
Inventor
郑宏伟
王歈
李双
张永华
李永
孙磊
李雪姣
Original Assignee
北京永泰生物制品有限公司
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Priority to PCT/CN2021/097191 priority Critical patent/WO2022252003A1/zh
Publication of WO2022252003A1 publication Critical patent/WO2022252003A1/zh

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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
    • C12M1/24—Apparatus for enzymology or microbiology tube or bottle type
    • 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
    • C12M1/26—Inoculator or sampler
    • 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
    • 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 invention relates to the technical field of cell culture, more specifically, to a closed culture system for separating, culturing and collecting peripheral blood mononuclear cells.
  • Mononuclear cells in peripheral blood refer to cells with a single nucleus in peripheral blood.
  • Mononuclear cells include two types of cells, one is lymphocytes and the other is monocytes.
  • lymphocytes There are two types of lymphocytes, B lymphocytes and T lymphocytes.
  • B lymphocytes have the function of humoral immunity. Under the stimulation of antigen, the morphology of B lymphocytes will change into plasma cells, and the plasma cells can produce and secrete antibodies, thereby exerting the function of humoral immunity; T lymphocytes have powerful cellular immunity functions, which can Directly kill pathogenic microorganisms or aging cells that enter the human body.
  • Monocytes are a type of human white blood cells.
  • mononuclear cells isolated and cultured from peripheral blood have the characteristics of fast proliferation, safety, persistence, adaptability, and systemicity. Compared with traditional radiotherapy and other methods, active immunization can stimulate systemic anti-tumor effects , with a wider scope of action, especially suitable for malignant tumors with multiple lesions or extensive metastases. Therefore, cultivating mononuclear cells isolated from peripheral blood and using them to treat diseases has become a trend in the medical field.
  • the existing mononuclear cell culture system is often very complicated to operate, especially when transferring liquid, it is necessary to repeatedly insert pipelines into each container, resulting in poor airtightness of the entire system, and the substances in the container are exposed to the outside world.
  • the risk of bacterial and virus contamination is high, so a highly sterilized operating space is required to reduce the risk of the material in the container being contaminated by external bacteria and viruses, which in turn makes the production process more complicated and costly.
  • the present invention proposes a closed culture system, which includes an inoculation room, a culture room, a liquid supplement room and a harvest room and a culture bottle suitable for transferring between them, wherein,
  • the inoculation room is provided with inoculation equipment, which is used to separate mononuclear cells from peripheral blood and transport the mononuclear cells to the culture flask;
  • the culture room is provided with culture equipment, which is used to accommodate the culture flask and provide the environment required for the culture of mononuclear cells;
  • the liquid replenishment room is provided with liquid replenishment equipment, which is used to deliver the medium required for the cultivation of mononuclear cells to the culture bottle;
  • the harvesting room is provided with a harvesting device for collecting mononuclear cells from the culture flask,
  • the culture bottle, the liquid replenishment equipment, and the harvesting equipment are all provided with pipelines with closed free ends, and the pipelines of the culture bottle are suitable for docking with the pipelines of the liquid replenishment equipment to form a connection between the two. and the pipeline of the culture bottle is also suitable for docking with the pipeline of the harvesting device to form a closed pipeline connecting the two.
  • the inoculation room is a sterile isolator.
  • the closed culture system further includes a sealable storage and transportation box, and the sealable storage and transportation box is used for accommodating and transferring the culture bottle.
  • the side wall of the sealable storage and transportation box is provided with an opening and a filter membrane covering the opening, the filter membrane is configured to allow water vapor and gas to pass through, and prevent the passage of microorganisms.
  • the inoculation room, the culture room, the fluid replacement room and the harvest room are all provided with sealable windows for the culture bottle to pass through.
  • the cultivation room is connected to the fluid replenishment room through a channel that is closed relative to the external environment, and the cultivation room and the harvesting room are connected through a channel that is closed to the external environment connected.
  • the culture equipment is provided with an airtight door suitable for closing its internal space, and the internal space is divided into a plurality of unit chambers isolated from each other, and each unit chamber Adapted to accommodate one culture bottle and provided with a unit door adapted to close it.
  • the rehydration equipment includes a sterile adapter, a peristaltic pump, and a rehydration bottle containing the culture medium
  • the rehydration bottle is provided with a pipeline with a closed free end
  • the aseptic adapter The machine is configured to connect the pipeline of the rehydration bottle with the pipeline of the culture bottle to form a closed pipeline
  • the peristaltic pump is configured to be installed on the closed pipeline.
  • the harvesting equipment includes an aseptic pipe-taking machine, a peristaltic pump, and a centrifuge cup
  • the centrifugal cup is provided with a pipeline with a closed free end
  • the aseptic pipe-taking machine is configured to
  • the pipeline of the centrifuge cup is docked with the pipeline of the culture bottle to form a closed pipeline
  • the peristaltic pump is configured to be installed on the closed pipeline.
  • the harvesting equipment further includes a centrifuge and an infusion bag
  • the centrifuge is used to rotate the centrifuge cup
  • the infusion bag is provided with a pipeline with a closed free end
  • the aseptic connecting machine is also configured to connect the pipeline of the centrifuge cup with the pipeline of the infusion bag to form another closed pipeline
  • the peristaltic pump is also configured to be installed in the another closed pipeline on the way.
  • Fig. 1 is a schematic layout diagram of each operation room of a closed culture system according to an embodiment of the present invention
  • FIG. 2 is a schematic perspective view of a culture bottle of a closed culture system according to an embodiment of the present invention
  • Fig. 3 is a schematic connection diagram of a culture bottle and a liquid replenishment device during a liquid replenishment operation in a closed culture system according to an embodiment of the present invention
  • Fig. 4 is a schematic connection diagram of a culture bottle and a harvesting device during a harvesting operation in a closed culture system according to an embodiment of the present invention.
  • Fig. 5 is a schematic perspective view of a closeable storage and transport box of a closed culture system according to an embodiment of the present invention.
  • the present invention aims to propose a closed culture system suitable for separating, cultivating and collecting high-purity peripheral blood mononuclear cells.
  • the closed culture system includes an inoculation room, a culture room, a fluid replacement room and a harvest room distributed on different stations. , culture bottles suitable for moving between various stations, and operating equipment and other consumables located in each operation room, wherein the inoculation room is equipped with a device capable of separating mononuclear cells from collected peripheral blood and dissolving mononuclear cells. Cells are transferred to the inoculation equipment in the culture bottle.
  • the culture room is equipped with culture equipment that can accommodate the culture bottle and provide the environment required for the cultivation of mononuclear cells.
  • the harvesting room is equipped with harvesting equipment that can collect the mononuclear cells in the culture bottle and transfer them to the infusion bag.
  • the blood collection bag containing the collected peripheral blood and the culture bottle containing the culture medium are first transferred to the inoculation room, and the mononuclear cells are separated from the peripheral blood by using the separator in the inoculation room and transferred to the inoculation room.
  • the culture bottle is transferred to the rehydration room In order to use the liquid replenishment equipment to replenish the medium in the culture bottle, and then transfer the culture bottle back to the culture equipment in the culture room to continue culturing the mononuclear cells. After a certain period of time (this period may include multiple After the culture of the first rehydration step), the culture flask was transferred to the harvesting room so that the mononuclear cells in the culture flask could be collected by harvesting equipment and transferred to the infusion bag.
  • the internal spaces of the inoculation room, the cultivation room, the rehydration room and the harvesting room are all isolated from the external environment, and can enter and exit through the sealable transfer window.
  • the culture operation, liquid rehydration operation and harvesting operation all provide a closed operating environment, which effectively reduces the risk of contamination of mononuclear cells by pollutants such as bacteria and viruses in the external environment, and also reduces the risk of mononuclear cells leaking to the external environment , which is conducive to the production of high-quality, pollution-free, high-purity mononuclear cells.
  • culture bottles and other consumables have their own pipelines, which only connect with other pipelines to form a closed pipeline when they need to transport substances, and are closed when they do not need to convey substances, which reduces the bacteria and viruses in the operating environment
  • the risk of contaminating mononuclear cells with other pollutants is reduced, and the risk of mononuclear cells leaking into the operating environment is also reduced, which further facilitates the production of high-quality, pollution-free, and high-purity mononuclear cells.
  • the airtight culture system comprises at least four operation rooms distributed on different stations, that is, an inoculation room 100, a culture room 200, a liquid replenishment room 300 and a harvest room 400, and a culture bottle that can move between each station 500.
  • FIG. 1 shows a schematic layout diagram of various operation rooms of the airtight culture system according to the present invention
  • FIG. 2 shows a schematic perspective view of a culture bottle 500 .
  • the culture bottle 500 includes a bottle body 510 suitable for containing culture medium and mononuclear cells and a bottle cap 520 connected to the bottle body 510, wherein the bottle cap 520 has a pipeline through which the Transport medium and mononuclear cells into and out of the bottle 510, so that the bottle cap 520 does not need to be removed from the bottle 510 when transporting the medium and mononuclear cells, which allows the bottle 510 to be manufactured integrally with the bottle cap 520 and
  • the culture bottle 500 is inseparable, and the risk of material in the bottle body 510 being leaked or contaminated due to opening the bottle cap 520 is reduced.
  • the free end of the pipeline on the bottle cap 520 is closed, and is only connected to other pipelines through a sterile adapter when it is necessary to transport culture medium and mononuclear cells, which further reduces the leakage of the material in the bottle body 510 or risk of contamination.
  • the inoculation room 100 accommodates the inoculation equipment and is provided with a sealable window
  • the inoculation equipment includes a separation device and an inoculation device
  • the sealable window is opened, the operator can pass through the sealable window to touch the inoculation equipment and move the culture bottle 500 in and out of the inoculation Room 100, and when the closeable window is closed, the inside of the inoculation room 100 is sealed relative to the outside.
  • the inoculation booth 100 may be provided with a sealable window for the operator to pass through and/or a sealable window for the culture bottle 500 to pass through.
  • the operator transfers the blood collection bag containing the collected peripheral blood and the culture bottle 500 containing the culture medium to the inoculation room 100 through the sealable window, and then uses the separation device to separate mononuclear cells from the peripheral blood , and then use the inoculation device to transfer the isolated mononuclear cells into the culture bottle 500, and finally move the culture bottle 500 containing the mononuclear cells and the culture medium out of the inoculation room 100 through the sealable window. More specifically, the bottle cap 520 of the culture bottle 500 can be removed first, then the mononuclear cells are transferred to the bottle body 510 of the culture bottle 500, and finally the bottle cap 520 is tightened with a torque wrench.
  • the bottle cap 520 can be screwed to a certain torque by a torque wrench, thereby achieving a suitable seal between the bottle body 510 and the bottle cap 520, so as to prevent mononuclear cells in the bottle body 510 from And the medium accidentally leaks or is polluted by external bacteria and viruses.
  • the mononuclear cells can also be transported to the bottle body 510 through the pipeline provided on the bottle cap 520 of the culture bottle 500, which further reduces the accidental leakage of the mononuclear cells and the culture medium in the bottle body 510 or contamination by external bacteria and viruses. risks, and also allows the use of the culture bottle 500 in which the bottle cap 520 is integrally manufactured with the bottle body 510 and cannot be separated.
  • the inoculation room 100 can be an aseptic isolator commonly used in the field, which is provided with a sealable window for the culture bottle 500 and the hand of the operator to enter and exit.
  • the culture room 200 accommodates culture equipment and is provided with a sealable window.
  • the culture equipment can be, for example, a carbon dioxide incubator, which can accommodate the culture bottle 500 and provide the culture requirements for mononuclear cells, such as by adjusting temperature, humidity, gas composition, etc. environment, and the sealable window allows the operator to touch the culture equipment and move the culture bottle 500 into and out of the culture room 200 through the sealable window when opened, and the inside of the culture room 200 is sealed relative to the outside when the sealable window is closed .
  • the culture room 200 may be provided with a sealable window for the operator to pass through and/or a sealable window for the culture bottle 500 to pass through.
  • the culture device can have an inner space for accommodating the culture bottle 500 and an airtight door that can close the inner space.
  • the inner space can be divided into a plurality of unit chambers isolated from each other, wherein each unit chamber One culture bottle 500 can be accommodated, and each unit chamber can also be provided with a separate unit door, so that a certain unit chamber can be individually closed.
  • the culture device can accommodate a plurality of culture flasks 500 so as to culture mononuclear cells in the plurality of culture flasks 500 at the same time. It is known that with the proliferation of mononuclear cells, the medium will be gradually consumed, so after the medium is consumed to a certain level, it is necessary to move the culture bottle 500 out of the culture equipment and the culture room 200 and move it into the rehydration room 300, In order to use the liquid replenishment equipment in the liquid replenishment room 300 to supplement the culture medium in the culture bottle 500 .
  • the rehydration room 300 accommodates a rehydration device 310 and is provided with a sealable window, the rehydration device 310 can supplement culture medium to the culture bottle 500, and the sealable window allows the operator to touch the rehydration device 310 and the rehydration device 310 through the sealable window when opened
  • the culture bottle 500 is moved in and out of the rehydration room 300, and the sealable window seals the interior of the rehydration room 300 from the outside when closed.
  • the rehydration room 300 may be provided with a sealable window for the operator to pass through and/or a sealable window for the culture bottle 500 to pass through. Referring to FIG.
  • the fluid replacement equipment 310 mainly includes a fluid replacement bottle 311, a peristaltic pump PP and a sterile adapter CD, wherein the fluid replacement bottle 311 contains a culture medium and has a pipeline, and the peristaltic pump PP can be installed on the top of the fluid replacement bottle 311 On the pipeline or be installed on the closed pipeline formed subsequently, in order to drive the culture medium in the rehydration bottle 311 to leave the rehydration bottle 311 and enter the culture bottle 500 through the pipeline, the aseptic adapter CD is configured for the pipeline of the rehydration bottle 311 It is connected with the pipeline of the culture bottle 500 so as to allow the culture medium leaving the rehydration bottle 311 to enter the culture bottle 500 , thereby supplementing the culture bottle 500 with the culture medium required for the cultivation of mononuclear cells.
  • the pipelines of the two will form a closed pipeline, and one end of the lumen of the closed pipeline is opened.
  • the other end leads to the culture bottle 500, and is isolated from the outside of the liquid rehydration bottle 311 and the culture bottle 500, that is to say, the liquid rehydration bottle 311 is connected to the culture bottle 500 through a closed pipeline, and the use of the peristaltic pump PP While making it possible to drive the culture medium through the closed pipeline, it also maintains the airtightness of the closed pipeline, so this configuration reduces the leakage of the culture medium in the rehydration bottle 311, the culture bottle 500 and the pipeline connecting the two to Risk of contamination from outside or by contaminants such as external bacteria and viruses, thus contributing to the production of high-purity, contamination-free, high-quality mononuclear cells.
  • this configuration also makes it necessary to use the aseptic adapter CD to connect the pipeline of the culture bottle 500 and the pipeline of the rehydration bottle 311 to carry out the rehydration operation, without temporarily inserting the tube into the rehydration bottle 311 or the culture bottle 500 This not only simplifies handling but also reduces the risk of contamination of the bottle contents by inserting tubing into each bottle.
  • the free end of the pipeline of the rehydration bottle 311 is also closed, and it is only connected to the pipeline of the culture bottle 500 through the aseptic adapter CD when the rehydration operation is required, which reduces the leakage of the culture medium in the rehydration bottle 311 There is no risk of external or contamination by external bacteria and viruses, which further contributes to the production of high-purity, pollution-free, high-quality mononuclear cells.
  • the operator moves the culture bottle 500 into the fluid replacement room 300 through the closable window, and then uses the aseptic adapter CD to connect the free end of the pipeline of the culture bottle 500 with the free end of the pipeline of the fluid replacement bottle 311, thereby forming A closed pipeline connecting the fluid supplement bottle 311 with the culture bottle 500 , after which a peristaltic pump PP is installed on the closed pipeline and the peristaltic pump PP is started to drive the culture medium in the fluid supplement bottle 311 into the culture bottle 500 .
  • the airtight pipeline can be cut off with thermal cutting pliers, and the free ends of the pipelines of the rehydration bottle 311 and the culture bottle 500 can be closed at the same time, and then the culture bottle 500 can be moved out of the rehydration room 300 through the sealable window and returned to the culture chamber. Room 200 and culture equipment to continue culturing mononuclear cells.
  • the culture bottle 500 needs to be moved out of the culture equipment and the culture room 200 and into the harvest room 400 so as to collect the mononuclear cells in the culture bottle 500 .
  • the harvesting room 400 accommodates the harvesting device 410 and is provided with a sealable window.
  • the harvesting device 410 can collect the culture in the culture bottle 500 and purify the mononuclear cells therefrom and deliver the purified mononuclear cells to the infusion bag, and the harvesting device 410 can
  • the closing window when open, allows an operator to access the harvesting device 410 and move the culture bottle 500 into and out of the harvesting chamber 400 through the closable window, while the closable window, when closed, seals the interior of the harvesting chamber 400 from the outside.
  • the harvesting chamber 400 may be provided with a sealable window for the passage of an operator and/or a sealable window for the passage of the culture bottle 500 .
  • FIG. 4 there is shown a schematic perspective view of a culture bottle 500 and a harvesting device 410 during a harvesting operation.
  • the harvesting device 410 mainly includes a centrifuge cup 411 , a centrifuge 412 , an infusion bag 413 , a sterile tube adapter CD and a peristaltic pump PP.
  • the centrifuge cup 411 is used to receive the culture from the culture bottle and deliver the purified mononuclear cells to the infusion bag 413 .
  • the centrifuge 412 is used to rotate the centrifuge cup 411 to stratify the culture in the centrifuge cup 411 so as to produce high-purity mononuclear cells.
  • the pipeline of culture bottle 500 is docked with the pipeline of centrifuge cup 411 by aseptic connecting machine CD, so that form the airtight pipeline that culture bottle 500 is connected with centrifuge cup 411 (the lumen of this airtight pipeline One end leads to the culture bottle 500, and the other end leads to the centrifuge cup 411, and is isolated from the outside of the culture bottle 500 and the centrifuge cup 411), then the peristaltic pump PP is set on the closed pipeline, and the peristaltic pump PP is started, so that the culture The culture in the bottle 500 is delivered to the centrifuge cup 411 through the airtight pipeline, and then the airtight pipeline is cut off with thermal cutting pliers, and the free ends of the pipelines of the culture bottle 500 and the centrifuge cup 411 are closed simultaneously.
  • the centrifuge cup 411 is installed in the centrifuge 412, and the centrifuge 412 is started to rotate the centrifuge cup 411, which makes the culture in the centrifuge cup 411 layered under the action of centrifugal force to produce high-purity mononuclear cells.
  • After centrifugal purification remove the centrifuge cup 411 from the centrifuge 412 and connect the pipeline of the centrifuge cup 411 with the pipeline of the infusion bag 413 by using the sterile adapter CD to form a connection between the centrifuge cup 411 and the infusion bag 413.
  • the centrifuge cup 411 Close the pipeline, then set the peristaltic pump PP on the closed pipeline, and start the peristaltic pump PP, so that the high-purity mononuclear cells in the centrifuge cup 411 are transported to the infusion bag 413, then, use thermal cutting forceps to place the The airtight pipeline is cut off, and the free ends of the pipelines of the centrifuge cup 411 and the infusion bag 413 are closed simultaneously. Finally, the infusion bag 413 containing highly purified mononuclear cells can be used for storage, transportation, treatment and other purposes. As mentioned above, under this configuration, the centrifuge cup 411 also has its own pipeline, and the free end of the pipeline is closed, and it is only connected to other pipelines when it is necessary to transport substances.
  • centrifugal cup 411 and the infusion bag 413 are all connected by a closed pipeline. Therefore, this configuration enables simple and convenient harvesting operations, and reduces the risk of the mononuclear cell cultures and extracts leaking to the outside or being contaminated by external bacteria, viruses, etc. during the harvesting operation, thereby contributing to the production of high-quality , pollution-free, high-purity mononuclear cells.
  • the closed culture system further includes a sealable storage and transportation box 600 for containing and transporting the culture bottle 500 .
  • the culture bottle 500 can be placed in the closable shipping box 600, and then the closable shipping box 600 can be closed, so that the culture can be transferred between various operations by transferring the closable shipping box 600 500 bottles.
  • the culture bottle 500 can be placed in the closable storage and transportation box 600 and the closable storage and transportation box 600 can be closed, and the closable storage and transportation box 600 can be transferred to the culture medium. Room 200.
  • the side wall of the closeable storage and transportation box 600 may be provided with an opening and a filter membrane covering the opening, and the filter membrane may allow water vapor and gas to pass through, but not allow microorganisms to pass through.
  • the closable storage and transportation box 600 allows the exchange of water vapor and gas between its interior and exterior, placing the culture bottle 500 in the closable storage and transportation box 600 will not affect the cultivation of mononuclear cells, thereby enabling The closeable storage and transport box 600 containing the culture bottle 500 is directly placed in the culture equipment for cultivation without taking out the culture bottle 500 from the sealable storage and transport box 600, which further simplifies the culture operation and reduces pollution and leakage risks of.
  • disinfection equipment may also be provided in the inoculation room 100, so as to sterilize the culture bottle 500 and the sealable storage and transport box 600 before and after the inoculation operation.
  • disinfection equipment can also be provided in other operating rooms, so as to sterilize the culture bottle 500 and the sealable storage and transport box 600 before and after corresponding operations.
  • closed passages P1, P2 and P3 connecting the cultivation room 200 with the inoculation room 100, the rehydration room 300 and the harvest room 400 can also be set, and the inside of the closed channels can be connected with the corresponding The internal connection between operations is connected, but it is isolated from the external between each operation.
  • one end of the airtight channel P1 is connected to the closable window of the inoculation room 100, and the other end is connected to the closable window of the culture room 200; and, one end of the airtight passage P3 is connected to the closable window of the cultivating room 200, and the other end is connected to the closable window of the harvesting room 400.
  • the operator in the inoculation room 100 can place the culture bottle 500 (or, the sealable storage box 600 containing the culture bottle 500 ) in the closed passage P1, and then place the culture bottle 500 in the culture room 200
  • the operator can take out the culture bottle 500 from the closed passage P1 and place it in the culture equipment, that is to say, the culture bottle 500 can be moved between each operation room through the respective closed passages P1, P2 and P3, so that Operators do not need to move the culture bottle 500 to enter and exit each operation room, which further improves the airtightness of the closed culture system, thereby further contributing to the production of high-quality, pollution-free and high-purity mononuclear cells.
  • the inoculation room 100 is an isolator, then there may be no closed passage P1, or the closed passage P1 may not be connected with the sealable window of the isolator, but only from the closeable window of the cultivation room 200. Protruding with a closeable free end.
  • the operator can take out the culture bottle 500 from the isolator after the inoculation operation is completed, and then put it through the free end of the closed passage P1 and place it in the closed passage P1, and then the operator in the cultivation room 200 can take it out from the closed passage P1.
  • the culture bottle 500 is removed from lane P1 and placed in the culture device. This configuration also eliminates the need for the operator to move the culture bottle 500 in and out of the various operating rooms.

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Abstract

本发明提供一种密闭培养系统，其包括接种间、培养间、补液间和收获间以及适合于在它们之间转移的培养瓶，其中，该接种间设有接种设备，其用于从外周血中分离出单个核细胞并将单个核细胞输送至培养瓶中；该培养间设有培养设备，其用于容纳培养瓶并提供单个核细胞的培养所需的环境；该补液间设有补液设备，其用于向培养瓶中输送单个核细胞的培养所需的培养基；该收获间设有收获设备，其用于从该培养瓶中收集单个核细胞，其中，该培养瓶、该补液设备和该收获设备均设有自由端封闭的管路，该培养瓶的管路适于与该补液设备的管路对接以形成将二者相连的密闭管路，并且该培养瓶的管路还适于与该收获设备的管路对接以形成将二者相连的密闭管路。

Description

密闭培养系统 技术领域
本发明涉及细胞培养技术领域，更具体地，涉及用于分离、培养和收集外周血单个核细胞的密闭培养系统。
背景技术
外周血是除骨髓之外的血液，外周血中的单个核细胞是指外周血中具有单个细胞核的细胞，单个核细胞包括两类细胞，一类是淋巴细胞，另一类是单核细胞。淋巴细胞又分成了两种，一种是B淋巴细胞，另一种是T淋巴细胞。B淋巴细胞具有体液免疫功能，在抗原刺激下，B淋巴细胞形态变形就会转变为浆细胞，浆细胞就可以产生分泌抗体，从而发挥体液免疫功能；T淋巴细胞具有强大的细胞免疫功能，可以直接杀死进入人体的病原微生物或者衰老的细胞。单核细胞是人体白细胞的一种，它从血液中逸出到组织就变成了吞噬细胞，可以吞噬衰老的细胞和细胞碎片，同时还能吞噬进入人体的各种病原微生物。而且，通过外周血分离培养的单个核细胞具有增殖速度快、安全性、持久性、适应性、全身性等特点，与传统的放疗等方法相比，通过主动免疫能够激发全身性的抗肿瘤效应，作用范围更加广泛，特别适用于多发病灶或有广泛转移的恶性肿瘤。因此，对从外周血中分离出来的单个核细胞进行培养，并利用其治疗疾病成为医疗领域中的一个趋势。然而，现有的单个核细胞的培养系统往往操作起来十分复杂，特别是在传输液体时需要向各个容器中反复插入管路，从而造成整个系统的密闭性较差，并且容器内的物质被外界细菌病毒污染的风险较高，为此则需要高度消毒杀菌的操作空间来降低容器内的物质被外界细菌病毒污染的风险，而这又造成生产流程更加复杂并且成本更加高昂。
因此，本领域中亟需一种操作起来简单方便并且具有较高的密闭性的培养措施。
发明内容
为了解决上述现有技术中的问题，本发明提出了一种密闭培养系统，其包括接种间、培养间、补液间和收获间以及适合于在它们之间转移的培 养瓶，其中，
所述接种间设有接种设备，其用于从外周血中分离出单个核细胞并将单个核细胞输送至所述培养瓶中；
所述培养间设有培养设备，其用于容纳所述培养瓶并提供单个核细胞的培养所需的环境；
所述补液间设有补液设备，其用于向所述培养瓶中输送单个核细胞的培养所需的培养基；
所述收获间设有收获设备，其用于从所述培养瓶中收集单个核细胞，
其中，所述培养瓶、所述补液设备和所述收获设备均设有自由端封闭的管路，所述培养瓶的管路适于与所述补液设备的管路对接以形成将二者相连的密闭管路，并且所述培养瓶的管路还适于与所述收获设备的管路对接以形成将二者相连的密闭管路。
根据本发明的一种可选的实施方式，所述接种间为无菌隔离器。
根据本发明的一种可选的实施方式，所述密闭培养系统还包括可封闭储运盒，所述可封闭储运盒用于容纳和转移所述培养瓶。
根据本发明的一种可选的实施方式，所述可封闭储运盒的侧壁设有开口以及将所述开口覆盖的滤膜，所述滤膜被配置成允许水蒸气和气体通过，并防止微生物通过。
根据本发明的一种可选的实施方式，所述接种间、所述培养间、所述补液间和所述收获间均设有供所述培养瓶通过的可封闭窗口。
根据本发明的一种可选的实施方式，所述培养间与所述补液间通过相对于外部环境封闭的通道相连接，并且所述培养间与所述收获间通过相对于外部环境封闭的通道相连接。
根据本发明的一种可选的实施方式，所述培养设备设有适于封闭其内部空间的气密门，所述内部空间被划分成彼此隔离的多个单元腔室，每个单元腔室适于容纳一个培养瓶并设有适于将其封闭的单元门。
根据本发明的一种可选的实施方式，所述补液设备包括无菌接管机、蠕动泵以及容纳培养基的补液瓶，所述补液瓶设有自由端封闭的管路，所述无菌接管机被配置成将所述补液瓶的管路与所述培养瓶的管路对接以形成密闭管路，所述蠕动泵被配置成安装在所述密闭管路上。
根据本发明的一种可选的实施方式，所述收获设备包括无菌接管机、蠕动泵以及离心杯，所述离心杯设有自由端封闭的管路，所述无菌接管机被配置成将所述离心杯的管路与所述培养瓶的管路对接以形成密闭管路，所述蠕动泵被配置成安装在所述密闭管路上。
根据本发明的一种可选的实施方式，所述收获设备还包括离心机和输液袋，所述离心机用于旋转所述离心杯，所述输液袋设有自由端封闭的管路，所述无菌接管机还被配置成将所述离心杯的管路与所述输液袋的管路对接以形成另一密闭管路，所述蠕动泵还被配置成安装在所述另一密闭管路上。
本发明可以体现为附图中的示意性的实施例。然而，应注意的是，附图仅仅是示意性的，任何在本发明的教导下所设想到的变化都应被视为包括在本发明的范围内。
附图说明
附图示出了本发明的示例性实施例。这些附图不应被解释为必然地限制本发明的范围，其中：
图1是根据本发明的一种实施方式的密闭培养系统的各个操作间的示意性布局图；
图2是根据本发明的一种实施方式的密闭培养系统的培养瓶的示意性立体图；
图3是根据本发明的一种实施方式的密闭培养系统在进行补液操作时的培养瓶和补液设备的示意性连接图；
图4是根据本发明的一种实施方式的密闭培养系统在进行收获操作时的培养瓶和收获设备的示意性连接图；以及
图5是根据本发明的一种实施方式的密闭培养系统的可封闭储运盒的示意性立体图。
具体实施方式
本发明的进一步的特征和优点将从以下参考附图进行的描述中变得更加明显。附图中示出了本发明的示例性实施例，并且各个附图并不必然地按照实际比例绘制。然而，本发明可以实现为许多不同的形式并且不应解 释为必然地限制于这里示出公开的示例性实施例。相反，这些示例性实施例仅仅被提供用于说明本发明以及向本领域的技术人员传递本发明的精神和实质。
本发明旨在提出一种适合于分离、培养、收集高纯度的外周血单个核细胞的密闭培养系统，该密闭培养系统包括分布在不同工位上的接种间、培养间、补液间和收获间、适合于在各个工位之间移动的培养瓶以及位于各个操作间中的操作设备和其他耗材，其中，接种间内设有可以从采集到的外周血中分离出单个核细胞并将单个核细胞转移至培养瓶中的接种设备，培养间内设有可以容纳培养瓶并提供单个核细胞的培养所需环境的培养设备，补液间中设有可以向培养瓶中补充培养基的补液设备，并且收获间中设有可以将培养瓶中的单个核细胞收集并转移至输液袋中的收获设备。在操作中，首先将含有采集到的外周血的采血袋以及含有培养基的培养瓶转移至接种间中，利用接种间中的分离器从外周血中分离出单个核细胞并将单个核细胞转移至培养瓶中，然后将培养瓶转移至培养间中的培养设备中以便对单个核细胞进行培养，在因单个核细胞的培养而消耗掉一定量的培养基之后，将培养瓶转移至补液间中以便利用补液设备向培养瓶中补充培养基，然后将培养瓶转移回培养间中的培养设备中以便继续对单个核细胞进行培养，在对单个核细胞进行了一定时期(该时期可能包含多个补液步骤)的培养之后，将培养瓶转移至收获间中以便利用收获设备将培养瓶中的单个核细胞收集并转移至输液袋中。需要指出的是，接种间、培养间、补液间和收获间的内部空间均相对于外部环境隔离开，并且可以通过可封闭的传递窗口进出，因此根据本发明的密闭培养系统为上述接种操作、培养操作、补液操作以及收获操作均提供了密闭的操作环境，这有效地降低了外部环境中的细菌病毒等污染物污染单个核细胞的风险，同时也降低了单个核细胞泄漏至外部环境的风险，从而有利于生产出高质量、无污染、高纯度的单个核细胞。另外，培养瓶以及其他耗材自带管路，该管路仅仅在需要输送物质时与其他管路对接从而形成密闭管路，而在不需要输送物质时封闭，这降低了操作环境中的细菌病毒等污染物污染单个核细胞的风险，同时也降低了单个核细胞泄漏至操作环境的风险，从而进一步有利于生产出高质量、无污染、高纯度的单个核细胞。
下面参考附图详细描述根据本发明的密闭培养系统的各个可选但非限制性的实施方式。
根据本发明的密闭培养系统包括分布在不同工位上的至少四个操作间，即，接种间100、培养间200、补液间300和收获间400以及可以在各个工位之间移动的培养瓶500。其中，图1示出了根据本发明的密闭培养系统的各个操作间的示意性布局图，图2示出了培养瓶500的示意性立体图。如图2所示，培养瓶500包括适于容纳培养基和单个核细胞的瓶体510和与瓶体510相连接的瓶盖520，其中，瓶盖520带有管路，通过该管路可以输送培养基和单个核细胞进出瓶体510，因此在输送培养基和单个核细胞时，不需要将瓶盖520从瓶体510上取下，这允许使用瓶体510与瓶盖520一体制造并且不可分离的培养瓶500，并且降低了因打开瓶盖520而造成瓶体510内的物质被泄漏或被污染的风险。特别地，瓶盖520上的管路的自由端封闭，并且仅仅在需要输送培养基和单个核细胞时通过无菌接管机与其他管路相连，这进一步降低了瓶体510内的物质被泄漏或被污染的风险。
接种间100容纳接种设备并且设有可封闭窗口，该接种设备包括分离装置和接种装置，并且该可封闭窗口在打开时允许操作人员穿过该可封闭窗口触及接种设备以及移动培养瓶500进出接种间100，而该可封闭窗口封闭时将接种间100的内部相对于外部密封。特别地，接种间100可以设有供操作人员通过的可封闭窗口和/或供培养瓶500通过的可封闭窗口。在操作时，操作人员将容纳有采集到的外周血的采血袋和容纳有培养基的培养瓶500通过可封闭窗口转移至接种间100中，然后利用分离装置从外周血中分离出单个核细胞，并进而利用接种装置将分离出的单个核细胞转移至培养瓶500中，最后将容纳单个核细胞以及培养基的培养瓶500通过可封闭窗口移出接种间100。更具体地，可以先取下培养瓶500的瓶盖520，然后将单个核细胞转移至培养瓶500的瓶体510中，最后用扭矩扳手拧紧瓶盖520。这种操作方式是有利的，因为可以通过扭矩扳手将瓶盖520旋拧至一定的扭矩，从而实现瓶体510与瓶盖520之间的合适的密封，以便防止瓶体510内的单个核细胞以及培养基意外泄漏或被外部细菌病毒等污染。当然，也可以通过培养瓶500的瓶盖520上设置的管路向瓶体510中输送单个核细胞，这进一步降低了瓶体510内的单个核细胞以及培养基意外泄 漏或被外部细菌病毒等污染的风险，并且还允许使用瓶盖520与瓶体510一体制造且不可分离的培养瓶500。特别地，接种间100可以是本领域中常用的无菌隔离器，其设有供培养瓶500以及操作人员的手进出的可封闭窗口。
培养间200容纳培养设备并且设有可封闭窗口，该培养设备例如可以是二氧化碳培养箱，其可以容纳培养瓶500并且例如通过调整温度、湿度、气体成分等等来提供单个核细胞的培养所需的环境，并且该可封闭窗口在打开时允许操作人员穿过该可封闭窗口触及培养设备以及移动培养瓶500进出培养间200，而该可封闭窗口封闭时将培养间200的内部相对于外部密封。特别地，培养间200可以设有供操作人员通过的可封闭窗口和/或供培养瓶500通过的可封闭窗口。在操作时，操作人员可以将容纳单个核细胞的培养瓶500穿过可封闭窗口移动至培养间200中，然后将培养瓶500置于培养设备中，该培养设备可以通过改变温度、湿度、气体成分等因素来提供单个核细胞的培养所需的环境。所述培养设备可以具有容纳培养瓶500的内部空间以及可以将该内部空间封闭的气密门，特别地，该内部空间可以被划分为彼此隔离的多个单元腔室，其中每个单元腔室可以容纳一个培养瓶500，并且每个单元腔室还可以设有单独的单元门，以便可以将某个单元腔室单独封闭。在该配置下，培养设备可以容纳多个培养瓶500，以便同时对多个培养瓶500内的单个核细胞进行培养。已知的是，随着单个核细胞的增殖，培养基会被逐渐消耗，因此在培养基消耗到一定水平之后，需要将培养瓶500移出培养设备和培养间200并将其移入补液间300，以便利用补液间300内的补液设备向培养瓶500内补充培养基。
补液间300容纳补液设备310并且设有可封闭窗口，该补液设备310可以向培养瓶500中补充培养基，并且该可封闭窗口在打开时允许操作人员穿过该可封闭窗口触及补液设备310以及移动培养瓶500进出补液间300，而该可封闭窗口封闭时将补液间300的内部相对于外部密封。特别地，补液间300可以设有供操作人员通过的可封闭窗口和/或供培养瓶500通过的可封闭窗口。参考图3，其中示出了补液操作时培养瓶500与补液设备310的示意性立体图。如图3所示，补液设备310主要包括补液瓶311、蠕动泵PP和无菌接管机CD，其中，补液瓶311容纳培养基并且带有管路， 蠕动泵PP可以被安装在补液瓶311的管路上或者被安装在后续形成的密闭管路上，以便驱动补液瓶311中的培养基通过管路离开补液瓶311进入培养瓶500，无菌接管机CD被配置用于将补液瓶311的管路与培养瓶500的管路相连接，以便允许离开补液瓶311的培养基进入培养瓶500中，从而向培养瓶500中补充单个核细胞的培养所需的培养基。在该配置下，在无菌接管机CD将补液瓶300的管路与培养瓶500的管路对接之后，二者的管路将形成一密闭管路，该密闭管路的管腔的一端通向补液瓶311，另一端通向培养瓶500，并且与补液瓶311和培养瓶500的外部隔离，也就是说，补液瓶311通过密闭管路与培养瓶500相连接，而且蠕动泵PP的使用在使得能够驱动培养基通过该密闭管路的同时也维持了该密闭管路的密闭性，因此该配置降低了补液瓶311、培养瓶500以及将二者相连的管路内的培养基泄漏至外部或被外部细菌病毒等污染物污染的风险，从而有助于生产出高纯度、无污染、高质量的单个核细胞。另外，该配置还使得仅仅需要利用无菌接管机CD将培养瓶500的管路与补液瓶311的管路对接既可进行补液操作，而不需要临时向补液瓶311或培养瓶500中插入管路，这不仅简化了操作而且也降低了因向各个瓶中插入管路而造成瓶内物质被污染的风险。特别地，补液瓶311的管路的自由端也是封闭的，并且仅仅在需要进行补液操作时通过无菌接管机CD与培养瓶500的管路对接，这降低了补液瓶311内的培养基泄漏之外部或被外部细菌病毒污染的风险，从而进一步有助于生产出高纯度、无污染、高质量的单个核细胞。在操作时，操作人员通过可封闭窗口将培养瓶500移入补液间300，然后利用无菌接管机CD将培养瓶500的管路的自由端与补液瓶311的管路的自由端对接，从而形成将补液瓶311与培养瓶500相连接的密闭管路，在此之后将蠕动泵PP安装在该密闭管路上并启动蠕动泵PP，以便将补液瓶311中的培养基驱动至培养瓶500中。在补液操作结束后，可以利用热切割钳将密闭管路切断，同时封闭补液瓶311和培养瓶500的管路的自由端，然后将培养瓶500通过可封闭窗口移出补液间300并移回培养间200和培养设备以便继续培养单个核细胞。
在完成培养操作之后，需要将培养瓶500移出培养设备和培养间200并将其移入收获间400，以便收集培养瓶500中的单个核细胞。收获间400 容纳收获设备410并且设有可封闭窗口，该收获设备410可以收集培养瓶500中的培养物并从中提纯单个核细胞并将提纯后的单个核细胞输送至输液袋中，而该可封闭窗口在打开时允许操作人员穿过该可封闭窗口触及收获设备410以及移动培养瓶500进出收获间400，而该可封闭窗口封闭时将收获间400的内部相对于外部密封。特别地，收获间400可以设有供操作人员通过的可封闭窗口和/或供培养瓶500通过的可封闭窗口。参考图4，其中示出了收获操作时培养瓶500与收获设备410的示意性立体图。如图4所示，收获设备410主要包括离心杯411、离心机412、输液袋413、无菌接管机CD以及蠕动泵PP。该离心杯411用于接收来自培养瓶的培养物以及向输液袋413输送提纯后的单个核细胞。该离心机412用于使离心杯411旋转从而使离心杯411中的培养物分层，以便产生高纯度的单个核细胞。在操作中，首先通过无菌接管机CD将培养瓶500的管路与离心杯411的管路对接，以便形成将培养瓶500与离心杯411相连的密闭管路(该密闭管路的管腔的一端通向培养瓶500，另一端通向离心杯411，并且与培养瓶500和离心杯411的外部隔离)，然后将蠕动泵PP设置在该密闭管路上，启动蠕动泵PP，以便将培养瓶500中的培养物通过该密闭管路输送至离心杯411中，然后利用热切割钳将该密闭管路切断，同时封闭培养瓶500和离心杯411的管路的自由端，在此之后，将离心杯411安装至离心机412中，启动离心机412以使离心杯411旋转，这使得离心杯411中的培养物将在离心力的作用下分层从而产生高纯度的单个核细胞，在进行离心提纯之后，从离心机412中取下离心杯411并利用无菌接管机CD将离心杯411的管路与输液袋413的管路相连接，从而形成将离心杯411与输液袋413相连的密闭管路，然后将蠕动泵PP设置在该密闭管路上，并启动蠕动泵PP，以便将离心杯411中的高纯度的单个核细胞输送至输液袋413中，然后，利用热切割钳将该密闭管路切断，同时封闭离心杯411和输液袋413的管路的自由端。最后，含有高纯度的单个核细胞的输液袋413可以用于存储、输运、治疗等目的。如上所述，在该配置下，离心杯411也自带管路，该管路的自由端封闭，并且仅仅在需要输送物质时与其他管路相连，而且，无论是培养瓶500与离心杯411还是离心杯411与输液袋413，都是通过密闭管路相连的。因此，该配置使得收获操作能够简单方便地进行， 而且降低了单个核细胞的培养物和提纯物在收获操作期间泄漏至外部或被外部细菌病毒等污染的风险，从而有助于生产出高质量、无污染、高纯度的单个核细胞。
可选地，如图5所示，根据本发明的密闭培养系统还包括可封闭储运盒600，该可封闭储运盒600用于容纳和转运培养瓶500。更具体地，可以将培养瓶500置于可封闭储运盒600中，然后将该可封闭储运盒600封闭，如此就可以通过转移可封闭储运盒600来在各个操作间之间转移培养瓶500。例如，可以在接种间100中的接种操作完成后，将培养瓶500置于可封闭储运盒600中并将该可封闭储运盒600封闭，并将该可封闭储运盒600转移至培养间200中。该配置是有利的，因为即使发生培养瓶500的瓶盖520没有拧紧、培养瓶500在转移过程中掉落等意外情况，培养瓶500中的物质也只会泄漏至可封闭储运盒600中，而不会泄漏至各个操作间中。特别地，可封闭储运盒600的侧壁可以设有开口以及覆盖所述开口的滤膜，该滤膜可以允许水蒸气和气体通过，但不允许微生物通过。在该配置下，由于可封闭储运盒600允许其内部和外部交换水蒸气和气体，因此将培养瓶500置于可封闭储运盒600中不会对单个核细胞的培养造成影响，从而可以直接将容纳有培养瓶500的可封闭储运盒600放在培养设备中进行培养，而无需从可封闭储运盒600中取出培养瓶500，这进一步简化了培养操作，而且降低了污染和泄漏的风险。
可选地，还可以在接种间100中设置消毒设备，以便在接种操作之前和之后对培养瓶500和可封闭储运盒600进行消毒杀菌。当然，还可以在其他操作间中设置消毒设备，以便在进行相应的操作之前和之后对培养瓶500和可封闭储运盒600进行消毒杀菌。
可选地，如图1所示，还可以设置将培养间200分别与接种间100、补液间300以及收获间400相连接的密闭通道P1、P2以及P3，所述密闭通道的内部可以与相应操作间的内部相连通，但相对于各个操作间的外部隔离。特别地，密闭通道P1一端连接至接种间100的可封闭窗口，另一端连接至培养间200的可封闭窗口；密闭通道P2一端连接至培养间200的可封闭窗口，另一端连接至补液间300的可封闭窗口；并且，密闭通道P3一端连接至培养间200的可封闭窗口，另一端连接至收获间400的可封闭窗口。 在该配置下，在接种操作完成后，接种间100内的操作人员可以将培养瓶500(或者，容纳培养瓶500的可封闭储运盒600)置于密闭通道P1中，然后培养间200内的操作人员可以从密闭通道P1中取出培养瓶500并将其置于培养设备中，也就是说，可以通过各个密闭通道P1、P2以及P3来在各个操作间之间移动培养瓶500，从而使得操作人员不必因需要移动培养瓶500而进出各个操作间，这进一步提高了本密闭培养系统的密闭性，从而进一步有助于生产出高质量、无污染、高纯度的单个核细胞。另外，需要指出的是，如果接种间100是隔离器，那么可能不存在密闭通道P1，或者密闭通道P1可能不与该隔离器的可封闭窗口连接，而是仅仅从培养间200的可封闭窗口突出并带有可封闭的自由端。在该情况下，操作人员可以在接种操作完成后从隔离器取出培养瓶500，然后将其穿过密闭通道P1的自由端置于密闭通道P1中，然后培养间200内的操作人员可以从密闭通道P1中取出培养瓶500并将其置于培养设备中。该配置同样使得操作人员不必因需要移动培养瓶500而进出各个操作间。
以上借助于附图详细描述了根据本发明的密闭培养系统的可选但非限制性的实施例。对于本领域内的那些普通技术人员来说，在不偏离本公开的精神和实质的情况下，对技术和结构的修改和补充以及对各实施例中的特征的重新组合显然都应视为包括在本发明的范围内。因此，在本发明的教导下所能够设想到的这些修改和补充都应被视为本发明的一部分。本发明的范围包括在本发明的申请日时已知的等效技术和尚未预见的等效技术。

Claims (10)

  1. 密闭培养系统，其包括接种间、培养间、补液间和收获间以及适合于在它们之间转移的培养瓶，其中，
    所述接种间设有接种设备，其用于从外周血中分离出单个核细胞并将单个核细胞输送至所述培养瓶中；
    所述培养间设有培养设备，其用于容纳所述培养瓶并提供单个核细胞的培养所需的环境；
    所述补液间设有补液设备，其用于向所述培养瓶中输送单个核细胞的培养所需的培养基；
    所述收获间设有收获设备，其用于从所述培养瓶中收集单个核细胞，
    其中，所述培养瓶、所述补液设备和所述收获设备均设有自由端封闭的管路，所述培养瓶的管路适于与所述补液设备的管路对接以形成将二者相连的密闭管路，并且所述培养瓶的管路还适于与所述收获设备的管路对接以形成将二者相连的密闭管路。
  2. 根据权利要求1所述的密闭培养系统，其中，所述接种间为无菌隔离器。
  3. 根据权利要求1所述的密闭培养系统，其中，所述密闭培养系统还包括可封闭储运盒，所述可封闭储运盒用于容纳和转移所述培养瓶。
  4. 根据权利要求3所述的密闭培养系统，其中，所述可封闭储运盒的侧壁设有开口以及将所述开口覆盖的滤膜，所述滤膜被配置成允许水蒸气和气体通过，并防止微生物通过。
  5. 根据权利要求1-4中任一项所述的密闭培养系统，其中，所述接种间、所述培养间、所述补液间和所述收获间均设有供所述培养瓶通过的可封闭窗口。
  6. 根据权利要求1-4中任一项所述的密闭培养系统，其中，所述培养间与所述补液间通过相对于外部环境封闭的通道相连接，并且所述培养间与所述收获间通过相对于外部环境封闭的通道相连接。
  7. 根据权利要求1-4中任一项所述的密闭培养系统，其中，所述培养设备设有适于封闭其内部空间的气密门，所述内部空间被划分成彼此隔离的多个单元腔室，每个单元腔室适于容纳一个培养瓶并设有适于将其封闭的单元门。
  8. 根据权利要求1-4中任一项所述的密闭培养系统，其中，所述补液设备包括无菌接管机、蠕动泵以及容纳培养基的补液瓶，所述补液瓶设有自由端封闭的管路，所述无菌接管机被配置成将所述补液瓶的管路与所述培养瓶的管路对接以形成密闭管路，所述蠕动泵被配置成安装在所述密闭管路上。
  9. 根据权利要求1-4中任一项所述的密闭培养系统，其中，所述收获设备包括无菌接管机、蠕动泵以及离心杯，所述离心杯设有自由端封闭的管路，所述无菌接管机被配置成将所述离心杯的管路与所述培养瓶的管路对接以形成密闭管路，所述蠕动泵被配置成安装在所述密闭管路上。
  10. 根据权利要求9所述的密闭培养系统，其中，所述收获设备还包括离心机和输液袋，所述离心机用于旋转所述离心杯，所述输液袋设有自由端封闭的管路，所述无菌接管机还被配置成将所述离心杯的管路与所述输液袋的管路对接以形成另一密闭管路，所述蠕动泵还被配置成安装在所述另一密闭管路上。
PCT/CN2021/097191 2021-05-31 2021-05-31 密闭培养系统 WO2022252003A1 (zh)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102061260A (zh) * 2010-12-01 2011-05-18 汪华 一种医用细胞自动化生产装置及其生产方法
CN107904175A (zh) * 2017-12-29 2018-04-13 广州沙艾生物科技有限公司 用于免疫细胞的诱导扩增的培养箱
CN109207424A (zh) * 2018-10-24 2019-01-15 河南省肿瘤医院 一种免疫细胞的培养方法
CN110835602A (zh) * 2018-08-18 2020-02-25 济南万泉生物技术有限公司 一种可持续灌流的三维细胞培养瓶
CN211394508U (zh) * 2019-11-06 2020-09-01 无锡生基医药科技有限公司 一种慢病毒载体全封闭生产的系统

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102061260A (zh) * 2010-12-01 2011-05-18 汪华 一种医用细胞自动化生产装置及其生产方法
CN107904175A (zh) * 2017-12-29 2018-04-13 广州沙艾生物科技有限公司 用于免疫细胞的诱导扩增的培养箱
CN110835602A (zh) * 2018-08-18 2020-02-25 济南万泉生物技术有限公司 一种可持续灌流的三维细胞培养瓶
CN109207424A (zh) * 2018-10-24 2019-01-15 河南省肿瘤医院 一种免疫细胞的培养方法
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