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WO2024139004A1 - 一种细胞自动化分选系统 - Google Patents

一种细胞自动化分选系统 Download PDF

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Publication number
WO2024139004A1
WO2024139004A1 PCT/CN2023/093281 CN2023093281W WO2024139004A1 WO 2024139004 A1 WO2024139004 A1 WO 2024139004A1 CN 2023093281 W CN2023093281 W CN 2023093281W WO 2024139004 A1 WO2024139004 A1 WO 2024139004A1
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WO
WIPO (PCT)
Prior art keywords
liquid
cell sorting
unit
buffer container
pipeline
Prior art date
Application number
PCT/CN2023/093281
Other languages
English (en)
French (fr)
Inventor
胡璧梁
尹果
谭亮
温磊
Original Assignee
楚天思为康基因科技(长沙)有限公司
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.)
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Application filed by 楚天思为康基因科技(长沙)有限公司 filed Critical 楚天思为康基因科技(长沙)有限公司
Publication of WO2024139004A1 publication Critical patent/WO2024139004A1/zh

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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
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors

Definitions

  • the utility model mainly relates to the technical field of medicine and food packaging machinery and equipment, and specifically relates to an automatic cell sorting system.
  • the technical problem to be solved by the utility model is: in view of the technical problems existing in the prior art, the utility model provides a cell automatic sorting system which can reduce the cell rupture rate.
  • the liquid inlet and the liquid outlet of the buffer container are both located at the bottom of the buffer container, and the air pump unit is communicated with the top of the buffer container.
  • the buffer container is a drip bucket.
  • the cell sorting unit comprises a plurality of cell sorting tubes, the inlets of the plurality of cell sorting tubes are all connected to the liquid outlet of the buffer container, and the outlets of the plurality of cell sorting tubes are all connected to the liquid collecting unit.
  • the connector assembly comprises a connector and a cap body, wherein the connector is located at the top of the cell sorting tube, and the cap body is covered on the connector.
  • the air pump unit is a peristaltic pump.
  • a bubble sensor is provided on the pipeline between the buffer container and the cell sorting unit.
  • the automatic cell sorting system further comprises a sealing detection unit for detecting the sealing of the pipeline between the liquid outlet of the liquid holding unit and the liquid inlet of the liquid collecting unit.
  • the airtightness detection unit includes a pressurizing component and a pressure detection component.
  • the pressurizing component is used to fill pressurized gas into the pipeline between the liquid outlet of the liquid holding unit and the liquid inlet of the liquid receiving unit;
  • the pressure detection component is used to detect the pressure of the pipeline between the liquid outlet of the liquid holding unit and the liquid inlet of the liquid receiving unit in a predetermined time period after pressurization.
  • the utility model discloses an automated cell sorting system, which arranges a buffer container between a liquid containing unit and a cell sorting unit, and then uses an air pump unit to evacuate and inflate the buffer container to control the inflow and discharge of liquid in the buffer container, so that the air pump unit can complete the transportation of cells without contacting the cells, thereby avoiding damage to the activity and morphology of the cells caused by the rollers in the pump, and reducing the cell rupture rate.
  • the liquid inlet and the liquid outlet of the buffer container are both located at the bottom of the buffer container, and the air pump unit is connected to the top of the buffer container, thereby reducing the generation of bubbles in the buffer container and further reducing the cell rupture rate.
  • FIG. 2 is a partial enlarged schematic diagram of portion A in FIG. 1 .
  • FIG3 is a schematic structural diagram of a sorting system according to an embodiment of the present invention.
  • FIG. 4 is a schematic structural diagram of the bottom connection port of the liquid-containing bag of the present invention in an embodiment.
  • FIG5 is a schematic structural diagram of the connection between the airtightness detection unit and the pipeline of the utility model.
  • FIG. 6 is a schematic structural diagram of an interface on a cell sorting tube according to an embodiment of the present invention.
  • Liquid holding unit 101. Cell liquid bag; 102. Buffer bag; 201-210. First to tenth pinch valves; 3. Air pump unit; 301. Peristaltic pump; 401. Bubble sensor; 5. Cell sorting unit; 501-503. First to third cell sorting tubes; 504.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as “first” or “second” may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of “plurality” is two or more, unless otherwise clearly and specifically defined.
  • the terms “assemble”, “connect”, “connect”, “fix” and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements.
  • the specific meanings of the above terms in this application can be understood according to specific circumstances.
  • the automatic cell sorting system of the embodiment of the utility model includes a liquid holding unit 1, a buffer container 7, an air pump unit 3, a cell sorting unit 5 and a liquid collecting unit 8.
  • the liquid holding unit 1 is connected to the liquid inlet of the buffer container 7, the liquid outlet of the buffer container 7 is connected to the liquid collecting unit 8 via the cell sorting unit 5, and the air pump unit 3 is connected to the buffer container 7 for evacuating or inflating the buffer container 7 to achieve the inflow and discharge of liquid in the buffer container 7.
  • the automatic cell sorting system of the present invention arranges a buffer container 7 between a liquid containing unit 1 and a cell sorting unit 5, and then evacuates and inflates the buffer container 7 through an air pump unit 3 to control the inflow and discharge of liquid in the buffer container 7, so that the air pump unit 3 can complete the transportation of cells without contacting the cells, thereby avoiding damage to the activity and morphology of the cells caused by the rollers in the pump and reducing the cell rupture rate.
  • Table 1 of Example 1 of the international patent application with the international application number "PCT/CN2023/092603" filed by the applicant on “May 6, 2023” and the invention title "A Cell Non-Destructive Delivery System” discloses the cell viability when the air pump unit 3 is in contact with the cells (the air pump unit 3 squeezes the cell fluid in the hose for delivery) and the cell viability when the air pump unit 3 is not in contact with the cells, proving that the method of squeezing the cell fluid in the hose for delivery by the air pump unit 3 will cause cell damage, resulting in a lower cell yield in this method than the cell yield in the method where the air pump unit 3 does not contact the cells but can complete the delivery of the cells.
  • This application adopts a technical concept similar to that of "PCT/CN2023/092603", that is, the air pump unit 3 does not contact the cells, and has similar technical effects.
  • the automated sorting system of the present application and the existing automated sorting system (Chinese patent application number 202221782866.9) were grouped and tested separately.
  • the cell viability of the existing automated sorting system (the air pump unit 3 squeezes the cell fluid in the hose for transportation) was significantly lower than the cell viability of the air pump unit 3 of the present application that does not contact the cells. It can be seen that the cell viability will be greatly reduced after the cells are transported by the air pump unit 3 (the air pump unit 3 squeezes the cell fluid in the hose for transportation).
  • the transportation method in which the cell fluid is not in direct contact with the air pump unit 3 has little effect on the cell viability. Therefore, the technical solution of this embodiment can be considered to cause no damage to the cells after the normal metabolism of the cells is discharged.
  • the liquid inlet and the liquid outlet of the buffer container 7 are both located at the bottom of the buffer container 7 , and the air pump unit 3 is connected to the top of the buffer container 7 .
  • the cell sorting unit 5 includes a plurality of cell sorting tubes, the inlets of the plurality of cell sorting tubes are connected to the liquid outlet of the buffer container 7, and the outlets of the plurality of cell sorting tubes are connected to the liquid collecting unit 8.
  • Each cell sorting tube is provided with a joint assembly 504 for injecting magnetic beads.
  • the joint assembly 504 includes a connector 5041 and a cap 5042, the connector 5041 is located at the top of the cell sorting tube, and the cap 5042 is covered on the connector 5041.
  • a tightness detection unit 9 is also included, which is used to detect the tightness of the pipeline between the liquid outlet of the liquid holding unit 1 and the liquid inlet of the liquid receiving unit 8.
  • the tightness detection unit 9 includes a pressurizing component and a pressure detection component.
  • the pressurizing component is used to fill the pipeline between the liquid outlet of the liquid holding unit 1 and the liquid inlet of the liquid receiving unit 8 with pressurized gas;
  • the pressure detection component is used to detect the pressure of the pipeline between the liquid outlet of the liquid holding unit 1 and the liquid inlet of the liquid receiving unit 8 for a predetermined period of time after pressurization.
  • the tightness detection unit 9 is used to perform a tightness detection on the sorting system to ensure the reliability of subsequent work.
  • a bubble sensor 401 is provided on the pipeline between the buffer container 7 and the cell sorting unit 5.
  • the bubble sensor 401 is used to detect whether there is liquid in the pipeline. When there is liquid, a feedback signal is given to the control system, and the system controls the peristaltic pump to start metering, providing accurate quantitative liquid addition for the subsequent pipeline.
  • liquid bags there are four liquid bags in total, including a cell liquid bag 101, a buffer bag 102, a cell collection liquid bag 801 and a waste liquid bag 802, which are made of transparent soft plastic materials that meet biocompatibility such as EVA, PVC or ULPDE.
  • connection ports at the bottom of the liquid bag, as shown in Figure 4. Specifically, a tube clamp, a 0.22 ⁇ m liquid filter (with a female connector) and a male cap are installed on the left connection port in sequence. The male cap can be removed in the biosafety cabinet, and a disposable sterile syringe with a male connector is used to add cell liquid samples, buffers, etc. to the liquid bag.
  • a tube clamp, a female connector and a male cap are installed on the middle connection port in sequence.
  • the male cap can be removed in the biosafety cabinet, and its female connector is connected to the male connector on the pipeline after the female cap is removed.
  • a tube clamp, a female connector and a male cap are installed on the right connection port in sequence.
  • the male cap can be removed in the biosafety cabinet, and its female connector is connected to the male connector on the pipeline after the female cap is removed.
  • the right port is a reserved interface.
  • the pipe is made of transparent plastic materials that meet biocompatibility, such as PVC or silicone tube;
  • the fourth pipeline 604 is connected to the first pipeline 601 and the second pipeline 602 through a T-shaped three-way connector.
  • the fourth pipeline 604 is connected to the bottom of the buffer drop bucket 701 (in this specific embodiment, the buffer drop bucket 701 is the buffer container 7), and the liquid is introduced from the bottom to reduce the generation of bubbles and avoid cell damage when the bubbles burst.
  • the two ends of the fifth pipeline 605 dedicated to the peristaltic pump are connected to the pipeline 605-A and the pipeline 605-B through the adapter, the pipeline 605-A is connected to the buffer drip bucket 701, and the pipeline 605-B is connected to the pipe clamp, 0.22 ⁇ m hydrophobic gas filter (with a female connector) and the male cap.
  • the peristaltic pump When in use, unscrew the male cap, the peristaltic pump reverses to pump air, so that the liquid in the cell liquid bag 101 and the buffer bag 102 enters the buffer drip bucket 701, and the peristaltic pump rotates forward to blow air and pressurize, so that the liquid entering the buffer drip bucket 701 is discharged and enters the subsequent pipeline 606.
  • the sixth pipeline 606 is connected to the seventh pipeline 607 , the eighth pipeline 608 , and the ninth pipeline 609 via a cross-shaped four-way connector.
  • the seventh pipeline 607, the eighth pipeline 608 and the ninth pipeline 609 are respectively provided with the third pinch valve 203, the fourth pinch valve 204 and the fifth pinch valve 205, which are respectively connected to the first cell sorting tube 501, the second cell sorting tube 502 and the third cell sorting tube 503, and then respectively connected to the tenth pipeline 610, the eleventh pipeline 611 and the twelfth pipeline 612.
  • the tenth pipeline 610 , the eleventh pipeline 611 , and the twelfth pipeline 612 are respectively provided with the sixth pinch valve 206 , the seventh pinch valve 207 , and the eighth pinch valve 208 , and are connected to the thirteenth pipeline 613 after being aggregated by a cross-shaped four-way joint.
  • the fourteenth pipeline 614 and the fifteenth pipeline 615 are respectively provided with a ninth pinch valve 209 and a tenth pinch valve 210.
  • the female caps on the fourteenth pipeline 614 and the fifteenth pipeline 615 can be removed in the biosafety cabinet, and their male connectors can be connected to the female connectors of the cell collection liquid bag 801 and the waste liquid bag 802 with the male caps removed to provide drainage for the pipeline system.
  • the first pinch valve 201 to the tenth pinch valve 210 are normally closed pinch valves.
  • the right interface is a connector assembly 504, which includes a connector 5041 and a cap body 5042.
  • the cap body 5042 can be removed in a biosafety cabinet, and the magnetic beads are injected into the cell sorting tube through a syringe, and then the cap body 5042 is installed and screwed tightly on the connector 5041.
  • the male caps on the left interfaces of the first cell sorting tube 501, the second cell sorting tube 502, and the third cell sorting tube 503 need to be installed and tightened; during testing, unscrew the male cap of the third pipeline 603, connect the 0.22 ⁇ m hydrophobic gas filter (with a female connector) to the leak detection pressurized pipeline, close the first pinch valve 201, the second pinch valve 202, the ninth pinch valve 209, and the tenth pinch valve 210, and open the third pinch valve 203, the fourth pinch valve 204, and the fifth pinch valve 2 05.
  • the sixth pinch valve 206, the seventh pinch valve 207, and the eighth pinch valve 208 are used to turn off the peristaltic pump, open the tube clamp of the third pipeline 603, and pressurize the pipeline.
  • stop pressurizing If there is no excessive pressure drop in the pipeline within the specified pressure time, the airtightness test is deemed to be qualified. After the test, disconnect the third pipeline 603 from the leak detection pressurized pipeline, let the pipeline system deflate, close the tube clamp, and tighten the male cap of the third pipeline 603.
  • the sorting system performs cell sorting or cell demagnetization in a closed system environment, without the need for a GMP environment, thus reducing the cost of maintaining the production environment.
  • the utility model forms a set of cell automated sorting consumables by rationally connecting and laying out the liquid bag, pipeline, clamp valve, peristaltic pump 301, bubble sensor 401 and sorting tube, thereby reducing the risk of contamination caused by human intervention in the development and production of cell therapy products.
  • the utility model designs a buffer drip bucket, and controls the inflow and discharge of liquid in the buffer drip bucket by pumping and inflating the buffer drip bucket, so that the pump can complete the cell delivery without contacting the cells, avoiding the damage to the cell activity and morphology caused by the roller in the pump.
  • the cell inlet and outlet of the buffer drip bucket are arranged at the bottom, and the pumping and blowing ports are arranged at the top, thereby reducing the generation of bubbles and reducing the damage to cells when the bubbles burst.
  • the cylindrical cell sorting tube of the utility model and the design of the top interface thereof can quickly add magnetic beads in a biological safety cabinet, and can also achieve pressure balance in the sorting tube when adding liquid.
  • the utility model can be used for sorting target cells in cell fluid after centrifugation, and can also be used for removing magnetic beads in cell fluid after sorting and enlarged culture.
  • the utility model can avoid the risk of contamination caused by human intervention in the development and production of cell therapy products, ensure the repeatability and consistency of cell sorting and demagnetization bead results, and meet the regulatory requirements for disposable, sterile, and airtight consumables for cell drug production.

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Abstract

本申请公开了一种细胞自动化分选系统，属于细胞自动化分选技术领域。该系统包括盛液单元(1)、缓冲容器(7)、气泵单元(3)、细胞分选单元(5)和收液单元(8)，盛液单元(1)与缓冲容器(7)的进液口相连，缓冲容器(7)的出液口经细胞分选单元(5)与收液单元(8)相连，气泵单元（3）与缓冲容器（7）连通，用于对缓冲容器（7）抽气或充气来实现缓冲容器（7）内液体的流入与排出。本申请通过在盛液单元与细胞分选单元之间布置缓冲容器，再通过气泵单元（3）对缓冲容器（7）进行抽气和充气作业，来控制缓冲容器（7）内的液体流入和排出，使气泵单元（3）不与细胞接触又能完成细胞的输送，避免泵内滚子碾压对细胞活性和形态的损伤，降低了细胞的破裂率。

Description

一种细胞自动化分选系统
相关申请的交叉引用
本申请以申请日为“2022年12月27日”、申请号为“202223602318.6”、发明创造名称为“一种细胞自动化分选系统”的中国专利申请为基础，并主张其优先权，该中国专利申请的全文在此引用至本申请中，以作为本申请的一部分。
技术领域
本实用新型主要涉及医药、食品包装机械设备技术领域，具体涉及一种细胞自动化分选系统。
 背景技术
目前国内外细胞治疗技术发展迅速，细胞治疗产品直接输入人体，无法进行终端灭菌或其他去除病毒的工艺处理。而在实验室、研发机构和相关生产中，常见细胞离心后直接将离心管在磁场中进行分选，再进行其它手动操作，容易对细胞样本产生污染，且市面上已有自动化分选设备的目的细胞得率和活性不高。
本专利申请人之前申请过关于细胞分选装置的专利申请（如中国专利申请号202221782866.9），来实现细胞的自动化分选以避免手动操作而对细胞样本的污染，但是上述专利申请对应的产品在测试以及使用过程中发现，分选出的细胞存在一定的破裂。经进一步深入的研究发现，其原因是泵直接对细胞液抽吸和排放，现有常规的蠕动泵挤压软管输送细胞液的示意图如图1和图2所示，进而导致细胞破裂。
 实用新型内容
本实用新型要解决的技术问题就在于：针对现有技术存在的技术问题，本实用新型提供一种降低细胞破裂率的细胞自动化分选系统。
为解决上述技术问题，本实用新型提出的技术方案为：
一种细胞自动化分选系统，包括盛液单元、缓冲容器、气泵单元、细胞分选单元和收液单元，所述盛液单元与所述缓冲容器的进液口相连，所述缓冲容器的出液口经所述细胞分选单元与所述收液单元相连，所述气泵单元与所述缓冲容器连通，用于对缓冲容器抽气或充气来实现缓冲容器内液体的流入与排出。
作为上述技术方案的进一步改进：
所述缓冲容器的进液口和出液口均位于所述缓冲容器的底部，所述气泵单元与所述缓冲容器的顶部连通。
所述缓冲容器为滴斗。
所述细胞分选单元包括多个细胞分选管，多个细胞分选管的入口均与缓冲容器的出液口相连，多个细胞分选管的出口均与收液单元相连。
各所述细胞分选管上设有用入注入磁珠的接头组件。
所述接头组件包括连接头和帽体，所述连接头位于所述细胞分选管的顶部，所述帽体盖设于所述连接头上。
所述气泵单元为蠕动泵。
所述缓冲容器与所述细胞分选单元之间的管路上设有气泡传感器。
所述细胞自动化分选系统还包括密闭性检测单元，用于检测盛液单元的出液口与收液单元的进液口之间管路的密封性。
所述密闭性检测单元包括加压组件和压力检测组件，所述加压组件用于向所述盛液单元的出液口与收液单元的进液口之间管路充入加压气体；所述压力检测组件用于检测所述盛液单元的出液口与收液单元的进液口之间管路在加压后预定时间段的压力。
与现有技术相比，本实用新型的优点在于：
本实用新型的细胞自动化分选系统，通过在盛液单元与细胞分选单元之间布置缓冲容器，再通过气泵单元对缓冲容器进行抽气和充气作业，来控制缓冲容器内的液体流入和排出，使气泵单元不与细胞接触又能完成细胞的输送，避免泵内滚子碾压对细胞活性和形态的损伤，降低细胞的破裂率。
本实用新型的细胞自动化分选系统，缓冲容器的进液口和出液口均位于缓冲容器的底部，而且气泵单元与缓冲容器的顶部连通，进而减少缓冲容器内气泡的产生，进一步降低细胞的破裂率。
 附图说明
图1是本发明背景技术中蠕动泵挤压软管输送细胞液的示意图。
图2是图1中A部分的局部放大示意图。
图3为本实用新型的分选系统在实施例的结构示意图。
图4为本实用新型的盛液袋的底部连接口在实施例的结构示意图。
图5为本实用新型的密闭性检测单元与管路连接的结构示意图。
图6为本实用新型的细胞分选管上的接口在实施例的结构示意图。
图例说明：1、盛液单元；101、细胞液袋；102、缓冲液袋；201～210、第一～第十夹管阀；3、气泵单元；301、蠕动泵；401、气泡传感器；5、细胞分选单元；501～503、第一～第三细胞分选管；504、接头组件；5041、连接头；5042、帽体；601～615、第一管路～第十五管路；7、缓冲容器；701、滴斗；8、收液单元；801、细胞收集液袋；802、废液袋；9、密闭性检测单元。
 具体实施方式
以下结合说明书附图和具体实施例对本实用新型作进一步描述。
在本申请的描述中，需要理解的是，术语 “上”、“下”、“左”、“右”、“前”、“后”、 “底”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本申请的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
在本申请中，除非另有明确的规定和限定，术语“装配”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本申请中的具体含义。
如图3所示，本实用新型实施例的细胞自动化分选系统，包括盛液单元1、缓冲容器7、气泵单元3、细胞分选单元5和收液单元8，盛液单元1与缓冲容器7的进液口相连，缓冲容器7的出液口经细胞分选单元5与收液单元8相连，气泵单元3与缓冲容器7连通，用于对缓冲容器7抽气或充气来实现缓冲容器7内液体的流入与排出。
本实用新型的细胞自动化分选系统，通过在盛液单元1与细胞分选单元5之间布置缓冲容器7，再通过气泵单元3对缓冲容器7进行抽气和充气作业，来控制缓冲容器7内的液体流入和排出，使气泵单元3不与细胞接触又能完成细胞的输送，避免泵内滚子碾压对细胞活性和形态的损伤，降低细胞的破裂率。
申请人于“2023年5月6日”递交的国际申请号为“PCT/CN2023/092603”，发明创造名称为“一种细胞无损输送系统”的国际专利申请实施例一的表一公开了气泵单元3与细胞接触（气泵单元3挤压软管内的细胞液进行输送）的细胞活率和气泵单元3不与细胞接触的细胞活率，证明气泵单元3挤压软管内的细胞液进行输送的方式会造成细胞损伤，导致该方式的细胞得率低于气泵单元3不与细胞接触又能完成细胞的输送方式的细胞得率。本申请采用与“PCT/CN2023/092603”类似的技术构思，即气泵单元3不与细胞接触，具有相似的技术效果。在采用相同原细胞液样本的情况下，将本申请的自动化分选系统和现有的自动化分选系统（中国专利申请号202221782866.9）分别分组进行试验，现有的自动化分选系统（气泵单元3挤压软管内的细胞液进行输送）的细胞活率明显低于本申请的气泵单元3不与细胞接触的细胞活率，由此可知，细胞经过气泵单元3输送后（气泵单元3挤压软管内的细胞液进行输送）的细胞活率会大幅减少，然而本实施例中，细胞液与气泵单元3未直接接触的这种输送方式对细胞活率影响不大，因此本实施例的技术方案在排出细胞正常的新陈代谢后可认为对细胞不产生损伤。
在一具体实施例中，缓冲容器7的进液口和出液口均位于缓冲容器7的底部，气泵单元3与缓冲容器7的顶部连通。
在一具体实施例中，细胞分选单元5包括多个细胞分选管，多个细胞分选管的入口均与缓冲容器7的出液口相连，多个细胞分选管的出口均与收液单元8相连。其中各细胞分选管上设有用入注入磁珠的接头组件504。具体地，接头组件504包括连接头5041和帽体5042，连接头5041位于细胞分选管的顶部，帽体5042盖设于连接头5041上。通过接头组件504的设计，可在生物安全柜中快速加入磁珠，也可实现加液时维持细胞分选管内的压力平衡。
在一具体实施例中，还包括密闭性检测单元9，用于检测盛液单元1的出液口与收液单元8的进液口之间管路的密封性。具体地，密闭性检测单元9包括加压组件和压力检测组件，加压组件用于向盛液单元1的出液口与收液单元8的进液口之间管路充入加压气体；压力检测组件用于检测盛液单元1的出液口与收液单元8的进液口之间管路在加压后预定时间段的压力。通过上述密闭性检测单元9对分选系统进行密闭性检测，保证后续工作的可靠性。
在一具体实施例中，缓冲容器7与细胞分选单元5之间的管路上设有气泡传感器401。气泡传感器401用于检测到管路内是否有液体，当有液体时反馈信号给控制系统，系统控制蠕动泵开始计量，为后续管路提供精准定量的加液。
下面结合附图来对本实用新型的分选系统的具体结构进行详细说明：
液袋设计：
共包括细胞液袋101、缓冲液袋102、细胞收集液袋801和废液袋802共四个液袋，由EVA、PVC或ULPDE等满足生物相容性透明软质塑料材质制成，液袋底部均有三个连接口，如图4所示。具体地，其中左侧连接口上依次安装管夹、0.22μm液体过滤器（带母连接头）和公帽，可在生物安全柜中取下公帽，采用带公连接头的一次性无菌注射器用于往液袋中加入细胞液样本、缓冲液等。中间连接口上依次安装管夹、母连接头和公帽，可在生物安全柜中取下公帽，将其母连接头与管路上取下母帽后的公连接头实现对接。右侧连接口上依次安装管夹、母连接头和公帽，可在生物安全柜中取下公帽，将其母连接头与管路上取下母帽后的公连接头实现对接。右侧口为预留接口。
管路设计：
管路由PVC或硅胶管等满足生物相容性的透明塑料材质制成；
其中第一管路601、第二管路602上分别设有第一夹管阀201、第二夹管阀202，可在生物安全柜中取下第一管路601、第二管路602上的母帽，将其公连接头与细胞液袋101、缓冲液袋102取下公帽后的母连接头实现对接，为管路系统提供加液。
其中第三管路603为第一管路601上的一条分叉管路，通过T字型三通接头相连。如图5所示，第三管路603上依次安装管夹、0.22μm疏水型气体过滤器（带母连接头）和公帽，可连接泄漏检测加压管路，用于密闭性检测。
其中第四管路604与第一管路601、第二管路602通过T字型三通接头相连。第四管路604连接缓冲滴斗701（在该具体实施例中，缓冲滴斗701即为缓冲容器7）的底部，从底部进液，可减少气泡产生，避免气泡破裂时对细胞损伤。
其中蠕动泵专用第五管路605两端通过转接头连接管路605-A和管路605-B，管路605-A连接缓冲滴斗701，管路605-B连接上依次安装管夹、0.22μm疏水型气体过滤器（带母连接头）和公帽。使用时，拧下公帽，蠕动泵反转抽气，使细胞液袋101、缓冲液袋102中的液体进入缓冲滴斗701，蠕动泵正转吹气加压，使进入缓冲滴斗701的液体排出，进入后续管路606。
其中第六管路606连接缓冲滴斗701的底部出液口，为后续管路供液，第六管路606设置有气泡传感器401，当气泡传感器401检测到管路内有液体时，反馈信号给控制系统，系统控制蠕动泵开始计量，为后续管路提供精准定量的加液。
其中第六管路606与第七管路607、第八管路608、第九管路609通过十字型四通接头相连。
其中第七管路607、第八管路608、第九管路609上分别设有第三夹管阀203、第四夹管阀204、第五夹管阀205，分别连接第一细胞分选管501、第二细胞分选管502、第三细胞分选管503后，在分别与第十管路610、第十一管路611、第十二管路612连接。
第十管路610、第十一管路611、第十二管路612上分别设有第六夹管阀206、第七夹管阀207、第八夹管阀208，经十字型四通接头汇总后，连接第十三管路613。
第十三管路613与第十四管路614、第十五管路615通过T字型三通接头相连。
第十四管路614、第十五管路615上分别设有第九夹管阀209、第十夹管阀210，可在生物安全柜中取下第十四管路614、第十五管路615上的母帽，将其公连接头与细胞收集液袋801、废液袋802取下公帽后的母连接头实现对接，为管路系统提供排液。
在一具体实施例中，第一夹管阀201至第十夹管阀210为常闭型夹管阀。
细胞分选管设计：
如图6所示，细胞分选管包括三个细胞分选管，记为第一细胞分选管501、第二细胞分选管502、第三细胞分选管503，第一细胞分选管501、第二细胞分选管502、第三细胞分选管503由PC或PS等满足多种生物相容性透明硬质塑料材质制成，分选管顶部均有两个接口。其中左侧接口上依次安装0.22μm疏水型气体过滤器（带母连接头）和公帽；生产使用前需取下公帽，用于加液时维持分选管内的压力平衡。右侧接口为接头组件504，包括连接头5041和帽体5042，可在生物安全柜中取下帽体5042，将磁珠通过注射器注入细胞分选管，然后将帽体5042安装旋紧密闭在连接头5041上。
管路系统泄漏检测说明：
检测前，第一细胞分选管501、第二细胞分选管502、第三细胞分选管503的左侧接口上的公帽需安装旋紧；检测时，旋开第三管路603的公帽，0.22μm疏水型气体过滤器（带母连接头）连接泄漏检测加压管路，关闭第一夹管阀201、第二夹管阀202、第九夹管阀209、第十夹管阀210，打开第三夹管阀203、第四夹管阀204、第五夹管阀205、第六夹管阀206、第七夹管阀207、第八夹管阀208，关闭蠕动泵，打开第三管路603的管夹，往管路中加压；当管路系统内部的压力到达检测限值时，停止加压；若在管路内的在压力规定时间内无超标的压降，则视为密闭性检测合格，检测后，断开第三管路603与泄漏检测加压管路的连接，让管路系统泄气后，关闭管夹，旋紧第三管路603的公帽。
使用案例说明：
上述分选系统既可用于离心后细胞液中的目的细胞分选，也可用于分选扩大培养后细胞液中的磁珠去除。
其中用于离心后细胞液中的目的细胞分选时，过程如下：
1）往第一细胞分选管501、第二细胞分选管502、第三细胞分选管503中加入磁珠，开启第二夹管阀202、第三夹管阀203、第四夹管阀204、第五夹管阀205，使用蠕动泵抽气使缓冲液袋102中的缓冲液进入缓冲滴斗701，再吹气加压进入各分选管进行清洗磁珠，磁铁靠近分选管，用磁铁吸附各分选管中磁珠后，开启第六夹管阀206、第七夹管阀207、第八夹管阀208、第十夹管阀210排出废液到废液袋802，关闭所有夹管阀，磁铁远离分选管；
2）开启第一夹管阀201、第三夹管阀203、第四夹管阀204、第五夹管阀205、蠕动泵301（在该具体实施例中，气泵单元3即为蠕动泵301）、气泡传感器401，使用蠕动泵301抽气使细胞液袋101中细胞液进入缓冲滴斗701，再吹气加压进入各分选管中，对分选管中的细胞和磁珠进行震荡混匀，使目的细胞与磁珠的充分结合孵育，再将磁铁靠近分选管，用磁铁吸附各分选管中目的细胞与磁珠的结合物后，开启第六夹管阀206、第七夹管阀207、第八夹管阀208、第十夹管阀210排出非目的细胞和其它杂质到废液袋802，关闭所有夹管阀，磁铁远离分选管；
3）开启第二夹管阀202、第三夹管阀203、第四夹管阀204、第五夹管阀205、蠕动泵301、气泡传感器401，使用蠕动泵301抽气使缓冲液袋102中缓冲液进入缓冲滴斗701，再吹气加压进入各分选管中，对分选管中的细胞进行震荡洗涤，磁铁靠近分选管，用磁铁吸附分选管中磁珠后，开启第六夹管阀206、第七夹管阀207、第八夹管阀208、第十夹管阀210排出非目的细胞和其它杂质到废液袋802，关闭所有夹管阀，磁铁远离分选管；
4)开启第二夹管阀202、第三夹管阀203、第四夹管阀204、第五夹管阀205、蠕动泵301、气泡传感器401，使用蠕动泵301抽气使缓冲液袋102中缓冲液进入缓冲滴斗701，再吹气加压进入各分选管中，对分选管中的细胞进行震荡重悬，开启第六夹管阀206、第七夹管阀207、第八夹管阀208、第九夹管阀209排出目的细胞到细胞收集液袋801；并再次重复一遍。
上述系统用于分选扩大培养后细胞液中的磁珠去除时，过程如下：
1)开启第一夹管阀201、第三夹管阀203、第四夹管阀204、第五夹管阀205、蠕动泵301、气泡传感器401，使用蠕动泵301抽气使细胞液袋101中细胞液进入缓冲滴斗701，再吹气加压进入各分选管中，对分选管中的液体进行震荡混匀，再将磁铁靠近分选管，用磁铁吸附分选管中细胞与磁珠的结合物后，开启第六夹管阀206、第七夹管阀207、第八夹管阀208、第九夹管阀209排出无磁珠的目的细胞到细胞收集液袋801，关闭所有夹管阀，磁铁远离分选管；
2)重复上述步骤a多次直至细胞液袋101中的细胞液处理完毕；
3)开启第二夹管阀202、第三夹管阀203、第四夹管阀204、第五夹管阀205、蠕动泵301、气泡传感器401，使用蠕动泵301抽气使缓冲液袋102中缓冲液进入缓冲滴斗701，再吹气加压进入分选管中，对分选管中细胞进行震荡洗涤，再将磁铁靠近分选管，用磁铁吸附分选管中目的细胞与磁珠的结合物后，开启第六夹管阀206、第七夹管阀207、第八夹管阀208、第九夹管阀209排出无磁珠的目的细胞到细胞收集液袋801，关闭所有夹管阀，磁铁远离分选管；
4)开启第二夹管阀202、第三夹管阀203、第四夹管阀204、第五夹管阀205、蠕动泵301、气泡传感器401，使用蠕动泵301抽气使缓冲液袋102中缓冲液进入缓冲滴斗701，再吹气加压进入分选管中，对分选管中细胞与磁珠的结合物进行震荡重悬，开启第六夹管阀206、第七夹管阀207、第八夹管阀208、第十夹管阀210排出细胞与磁珠的结合物到废液袋802。
其它说明：
分选系统中细胞分选管及其前后夹管阀的数量可根据生产使用量的需要进行调整；
分选系统进行细胞分选或细胞去磁珠，均在封闭的系统环境中进行，无需提供GMP环境，降低了生产环境的维持成本；
分选系统的液袋与管路可直接接好，形成一套完整的耗材进行灭菌和包装处理；也可将液袋与管路分开进行灭菌和包装处理，使用时在生物安全柜中通过公母对接头接好，然后进行实验或生产。
分选系统为一次性使用产品，需保证无菌条件，进行环氧乙烷灭菌或伽马射线辐照灭菌，并采用医用级灭菌包装袋进行包装；包装袋上有二维识别码，使用时采用扫码枪进行扫码录入操作，记录耗材信息；使用后可对管路进行热合封闭，再按医疗废弃物标准进行无公害处理，防止泄漏污染环境。
本实用新型通过对液袋、管路、夹管阀、蠕动泵301、气泡传感器401和分选管等进行合理地连接布局设计，组成一套细胞自动化分选耗材，减少细胞治疗产品研发生产过程中人为干预带来的污染风险。
本实用新型设计缓冲滴斗，通过泵对缓冲滴斗抽气和充气来控制缓冲滴斗内的液体流入和排出，使泵不与细胞接触又能完成细胞的输送，避免了泵内滚子碾压对细胞活性和形态的损伤。其中缓冲滴斗的细胞进口和出口设置在底部，泵的抽、吹气口设置在顶部，进而减少气泡的产生，降低气泡破裂时对细胞损伤。
本实用新型的圆柱形细胞分选管及其顶部接口的设计，可在生物安全柜中快速加入磁珠，也可实现加液时维持分选管内的压力平衡。
本实用新型既可用于离心后细胞液中的目的细胞分选，也可用于分选扩大培养后细胞液中的磁珠去除。
本实用新型可避免细胞治疗产品研发、生产过程中人为干预带来的污染风险，保证细胞分选和去磁珠结果的重复性一致性，满足法规对细胞药物生产的一次性、无菌性、密闭性耗材的要求。
以上仅是本实用新型的优选实施方式，本实用新型的保护范围并不仅局限于上述实施例，凡属于本实用新型思路下的技术方案均属于本实用新型的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本实用新型原理前提下的若干改进和润饰，应视为本实用新型的保护范围。。

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  1.  一种细胞自动化分选系统，其特征在于，包括盛液单元(1)、缓冲容器(7)、气泵单元(3)、细胞分选单元(5)和收液单元(8)，所述盛液单元(1)与所述缓冲容器(7)的进液口相连，所述缓冲容器(7)的出液口经所述细胞分选单元(5)与所述收液单元(8)相连，所述气泵单元(3)与所述缓冲容器(7)连通，用于对缓冲容器(7)抽气或充气来实现缓冲容器(7)内液体的流入与排出。
  2.  根据权利要求1所述的细胞自动化分选系统，其特征在于，所述缓冲容器(7)的进液口和出液口均位于所述缓冲容器(7)的底部，所述气泵单元(3)与所述缓冲容器(7)的顶部连通。
  3.  根据权利要求1所述的细胞自动化分选系统，其特征在于，所述缓冲容器(7)为滴斗(701)。
  4.  根据权利要求1或2或3所述的细胞自动化分选系统，其特征在于，所述细胞分选单元(5)包括多个细胞分选管，多个细胞分选管的入口均与缓冲容器(7)的出液口相连，多个细胞分选管的出口均与收液单元(8)相连。
  5.  根据权利要求4所述的细胞自动化分选系统，其特征在于，各所述细胞分选管上设有用入注入磁珠的接头组件(504)。
  6.  根据权利要求5所述的细胞自动化分选系统，其特征在于，所述接头组件(504)包括连接头(5041)和帽体(5042)，所述连接头(5041)位于所述细胞分选管的顶部，所述帽体(5042)盖设于所述连接头(5041)上。
  7.  根据权利要求1或2或3所述的细胞自动化分选系统，其特征在于，所述气泵单元(3)为蠕动泵(301)。
  8.  根据权利要求1或2或3所述的细胞自动化分选系统，其特征在于，所述缓冲容器(7)与所述细胞分选单元(5)之间的管路上设有气泡传感器(401)。
  9.  根据权利要求1或2或3所述的细胞自动化分选系统，其特征在于，所述细胞自动化分选系统还包括密闭性检测单元(9)，用于检测盛液单元(1)的出液口与收液单元(8)的进液口之间管路的密封性。
  10.  根据权利要求9所述的细胞自动化分选系统，其特征在于，所述密闭性检测单元(9)包括加压组件和压力检测组件，所述加压组件用于向所述盛液单元(1)的出液口与收液单元(8)的进液口之间管路充入加压气体；所述压力检测组件用于检测所述盛液单元(1)的出液口与收液单元(8)的进液口之间管路在加压后预定时间段的压力。
PCT/CN2023/093281 2022-12-27 2023-05-10 一种细胞自动化分选系统 WO2024139004A1 (zh)

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