Cell culture method

Abstract

The invention provides a cell culture method which comprises the following steps: S100, carrying out enlarged culture on to-be-cultured cells, and transferring the to-be-cultured cells into a two-layer cell factory for cell culture; s200, transferring the cells cultured in the two-layer cell factory into a 10-layer cell factory under the assistance of a mechanical arm, and culturing; s300, transferring the cells cultured in the 10-layer cell factory into a 40-layer cell factory under the assistance of a mechanical arm, and culturing; and S400, loading 40 layers of cells cultured in the cell factory into a third liquid storage vehicle under the assistance of a mechanical arm for later use. According to the invention, through combination of an automation technology and gradual amplification culture of a cell factory, a plurality of challenges, including pollution risk, operation complexity and expandability problems, faced in traditional cell culture are effectively solved, so that a more efficient and more reliable solution is provided for large-scale biological product production.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a cell culture method. Background Art

[0002] Cell culture refers to a technical method that supports cell growth, reproduction, and maintenance of cell function and structure by regulating appropriate temperature, pH, and essential nutrients under a sterile environment. In the biopharmaceutical field, cell factories are a commonly used cell culture tool, widely used in cell expansion and large-scale production processes, particularly in the production of vaccines, monoclonal antibodies, and other biological products. Common cell factory specifications include one-layer, two-layer, 10-layer, and 40-layer structures, which are used for large-scale cell culture to meet the needs of industrial production. During cell expansion, multiple cell factories are often required to operate in parallel to ensure consistency and stability in cell density and growth during large-scale culture. To achieve this goal, multiple cell factories are often connected in series and cultured together in a cell factory incubator through a unified packaging and culture process. However, operating multiple cell factories for large-scale cell culture often involves complex operational procedures, and frequent open-cell operations increase the risk of contamination, which may affect cell culture stability and product quality. Summary of the Invention

[0003] In view of this, the present invention provides a cell culture method, which is convenient to operate, has a low risk of contamination, and improves the stability of cell culture and the quality of the product.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a cell culture method, comprising the following steps: S100, after the cells to be cultured are expanded and cultured, they are transferred to the second-layer cell factory for cell culture; S200, the cells cultured in the 2-layer cell factory are transferred to the 10-layer cell factory for culture with the assistance of a robotic arm; S300: The cells cultured in the 10-layer cell factory are transferred to the 40-layer cell factory for culture with the assistance of a robotic arm; S400: The cells cultured in the 40-layer cell factory are loaded into the third liquid storage vehicle with the assistance of a robotic arm for standby use.

[0005] Preferably, in step S100, the specific steps of expanding the cells to be cultured are: thawing and expanding the cells to be cultured in a T75 flask, and then digesting and transferring them into a two-layer cell factory for cell culture.

[0006] Preferably, in step S200, the specific steps of transferring the cells cultured in the 2-layer cell factory to the 10-layer cell factory for culture with the assistance of a robotic arm are as follows: S210, digesting the cells cultured in the two-layer cell factory and then loading them into the first liquid storage vehicle; S220, connecting the first liquid storage vehicle to the 10-layer cell factory with the assistance of a robotic arm, so that the cell solution in the first liquid storage vehicle enters each layer of the 10-layer cell factory; S230: After separating the first liquid storage vehicle from the 10-layer cell factory with the assistance of a robotic arm, the 10-layer cell factory is pushed to a vehicle positioning system and then to a cell culture room for culture.

[0007] Preferably, the number of the 10-layer cell factories is 2, and the two 10-layer cell factories are interconnected.

[0008] Preferably, in step S300, the cells cultured in the 10-layer cell factory are mechanically The specific steps for arm-assisted transfer into a 40-layer cell factory for culture are: S310, place each 10-layer cell factory under a cell factory microscope to observe the culture conditions. After the culture conditions are met, the 10-layer cell factory is moved to the liquid adding platform; S320, adding digestion fluid to the 10-layer cell factory with the assistance of a robotic arm, and completing Digestion; S330, connect the second liquid storage vehicle to the 10-layer cell factory through a robotic arm, so that The digested cells in the 10-layer cell factory enter the second liquid storage vehicle; S340: After the second liquid storage vehicle is separated from the 10-layer cell factory by a robotic arm, connecting the second liquid storage vehicle to the 40-layer cell factory to transport the digested cells in the 40-layer cell factory to each layer of the 40-layer cell factory; S350, separate the second liquid storage vehicle from the 40-layer cell factory with the assistance of a robotic arm After opening, the 40-layer cell factory is pushed to the trolley positioning system and then to the cell culture room for culture.

[0009] Preferably, in step S320, digestion fluid is added to the 10-layer cell factory with the assistance of a robotic arm, and the specific steps for completing digestion are: pour out the old cell culture fluid, rotate the 10-layer cell factory to the liquid adding position, add cell digestion fluid for the first time to wash the cell surface, discard the cell digestion fluid, and rotate the 10-layer cell factory group to the liquid adding position; add cell digestion fluid to infiltrate the cell surface, place it on the cell factory group frame, and enter the cell digestion countdown; the robotic arm grabs the 10-layer cell factory, rotates 90° to observe the cell digestion situation, adds cell culture medium to terminate digestion, homogenizes the liquid, and oscillates it with a small amplitude and shakes it quickly with the assistance of the robotic arm; homogenizes the liquid again with the assistance of the robotic arm with a small amplitude and quickly oscillates to complete the digestion.

[0010] Preferably, in step S400, the cells cultured in the 40-layer cell factory are loaded into the third liquid storage vehicle with the assistance of a robotic arm, and the specific steps for use are as follows: S410, placing each 40-layer cell factory under a cell factory microscope to observe the culture conditions. After the culture conditions are met, the 40-layer cell factory is moved to a liquid addition platform; S420, adding digestion fluid to the 40-layer cell factory with the assistance of a robotic arm to complete digestion; S430, connecting the third liquid storage vehicle to the 40-layer cell factory via a robotic arm, so that the digested cells in the 40-layer cell factory enter the third liquid storage vehicle.

[0011] Preferably, in step S420, digestion fluid is added to the 40-layer cell factory with the assistance of a robotic arm, and the specific steps for completing digestion are: pour out the old cell culture fluid, rotate the 40-layer cell factory to the liquid adding position, add cell digestion fluid for the first time to wash the cell surface, discard the cell digestion fluid, and rotate the 40-layer cell factory group to the liquid adding position; add cell digestion fluid to infiltrate the cell surface, place it on the cell factory group frame, and enter the cell digestion countdown; the robotic arm grabs the 40-layer cell factory, rotates 90° to observe the cell digestion situation, adds cell culture medium to terminate digestion, homogenizes the liquid, and oscillates it with a small amplitude and shakes it quickly with the assistance of the robotic arm; homogenizes the liquid again with the assistance of the robotic arm with a small amplitude and quick oscillation to complete the digestion.

[0012] Preferably, the number of the 40-layer cell factories is 4, and the 4 40-layer cell factories are interconnected.

[0013] Preferably, the 2-layer cell factory, the 10-layer cell factory and the 40-layer cell factory all include a liquid inlet and a liquid outlet, the liquid inlet is used to input liquid, and the liquid outlet is used to discharge liquid.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention effectively solves multiple challenges faced in traditional cell culture, including contamination risk, operational complexity and scalability issues, by combining automation technology and the gradual expansion of cell factory groups, thereby providing a more efficient and reliable solution for large-scale production of biological products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A flow chart of a cell culture method provided in one embodiment of the present invention; Figure 2 A flow chart of transferring cells cultured in a 2-layer cell factory to a 10-layer cell factory for culture with the assistance of a robotic arm, according to one embodiment of the present invention; Figure 3 This is a flow chart of transferring cells cultured in a 10-layer cell factory to a 40-layer cell factory for culture with the assistance of a robotic arm according to one embodiment of the present invention; Figure 4 A flow chart of loading cells cultured in a 40-layer cell factory into a third liquid storage vehicle for standby use with the assistance of a robotic arm according to one embodiment of the present invention; Figure 5 A diagram showing the connection between the two-layer cell factory and the first liquid storage vehicle provided in one embodiment of the present invention; Figure 6 A connection diagram of a 10-layer cell factory group and a first liquid storage vehicle provided in one embodiment of the present invention; Figure 7 A diagram showing the connection between the 10-layer cell factory group, each liquid storage vehicle, and the second liquid storage vehicle according to one embodiment of the present invention; Figure 8 This is a connection diagram between the 40-layer cell factory group and each liquid storage vehicle and the third liquid storage vehicle provided in one embodiment of the present invention.

[0016] Reference numerals: 2-layer cell factory 1 , first liquid storage vehicle 2 , 10-layer cell factory 3 , second liquid storage vehicle 4 , 40-layer cell factory 5 , various liquid storage vehicles 6 , third liquid storage vehicle 7 .

[0017] Note: The liquid storage vehicles 6 mentioned throughout the text do not include the second liquid storage vehicle 4 and the third liquid storage vehicle 7. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0019] Cell culture refers to a technical method that supports cell growth, reproduction, and maintenance of cell function and structure by regulating appropriate temperature, pH, and essential nutrients under a sterile environment. In the biopharmaceutical field, cell factories are a commonly used cell culture tool, widely used in cell expansion and large-scale production processes, particularly in the production of vaccines, monoclonal antibodies, and other biological products. Common cell factory specifications include one-layer, two-layer, 10-layer, and 40-layer structures, which are used for large-scale cell culture to meet the needs of industrial production. During cell expansion, multiple cell factories are often required to operate in parallel to ensure consistency and stability in cell density and growth during large-scale culture. To achieve this goal, multiple cell factories are often connected in series and cultured together in a cell factory incubator through a unified packaging and culture process. However, operating multiple cell factories for large-scale cell culture often involves complex operational procedures, and frequent open-cell operations increase the risk of contamination, which may affect cell culture stability and product quality.

[0020] In order to solve the above problems, combined Figure 1 The present invention provides a cell culture method comprising the following steps: S100, after the cells to be cultured are expanded and cultured, they are transferred to the second layer of cell factory 1 for cell culture. cell culture; Specifically, the cells to be cultured are not limited and can be used to culture various cells, such as MDCK, Vero, and 293T, to achieve automated cell passage. In the prior art, the structure of a cell factory primarily consists of a multi-layer culture surface, a culture medium and gas supply system, a temperature and pH control system, a sterile filtration system, and a monitoring and control system. It is designed to provide a stable, controllable, and efficient environment for cell growth and proliferation, meeting the needs of industrialized production of biological products. The following cell factories all share this feature.

[0021] S200, the cells cultured in the 2nd layer of cell factory 1 are transferred to the 10th layer with the assistance of a robotic arm Cell factory 3 is cultured; S300, the cells cultured in the 10-layer cell factory 3 are transferred to the 40-layer cell factory with the assistance of a robotic arm. Cell factory 5 is cultured; S400: The cells cultured in the 40-layer cell factory 5 are loaded into the third storage liquid with the assistance of a robotic arm. Car 7 is in the middle and is on standby.

[0022] In the above technical solution, the present invention can better control the growth state of cells through the gradual expansion process of 2 layers, 10 layers to 40 layers, ensuring that cells can obtain appropriate growth space and nutrient supply at each stage. The present invention uses a robotic arm to perform cell transfer operations, which can greatly reduce the risk of contamination caused by manual operation and improve the sterility of the culture process. The present invention does not limit the specific structure of the robotic arm, as long as it can be controlled by a computer or other port to replace manual work; the robotic arm-assisted transfer can improve operational efficiency, reduce manual operation time, and speed up the entire culture process. In short, this cell culture method effectively solves the problems of operational complexity and contamination risk in large-scale cell culture by combining mechanized operation and staged expansion culture, while improving culture efficiency and product quality, which is of great significance to industrial production in the field of biomedicine.

[0023] Furthermore, in step S100, the specific steps of expanding the culture of the cells to be cultured are as follows: the cells to be cultured are recovered and expanded in a T75 flask, and then digested and transferred to the 2-layer cell factory 1 for cell culture. In this technical solution, cell recovery is to thaw the frozen cells to be cultured and place them in a T75 culture flask for recovery. The T75 culture flask is a commonly used laboratory-grade cell culture container with a 75 cm 2 Growth area. Initial expansion culture is to expand the culture of cells in a T75 flask. This process usually includes regular replacement of culture medium, observation of cell growth status, and subculturing when the cells reach a certain density. Cell digestion is when the cells grow to an appropriate density, using digestive enzymes (such as trypsin) to peel the cells from the bottom of the T75 flask. This step transforms the cells from a monolayer adherent state to a suspended state, facilitating subsequent transfer. Transfer to the 2-layer cell factory 1 is to transfer the digested cell suspension to the 2-layer cell factory 1 for continued culture. The 2-layer cell factory 1 provides a larger growth area, which is conducive to further cell expansion.

[0024] Combine Figure 2 Furthermore, in step S200, the cells cultured in the 2-layer cell factory 1 are transferred to the 10-layer cell factory 3 for culture with the assistance of a robotic arm. The specific steps are: S210, digesting the cells cultured in the two-layer cell factory 1 and then loading them into the first liquid storage vehicle 2; In some embodiments, illustratively, the cell suspension of the digested two-layer cell factory 1 is peristaltically transferred to the first liquid storage vehicle 2 and shaken; S220, connecting the first liquid storage vehicle 2 to the ten-layer cell factory 3 with the assistance of a robotic arm, so that the cell solution in the first liquid storage vehicle 2 enters each layer of the ten-layer cell factory 3; In some embodiments, illustratively, the robotic arm operates to divide the cell suspension into 10-layer cell factories 3. It should be noted that the number of 10-layer cell factories 3 of the present invention is not specifically limited, and can be 1, 2 or even more. When the number of 10-layer cell factories 3 is more than one, multiple 10-layer cell factories 3 can be regarded as a factory group. For example, when the number of 10-layer cell factories 3 is 2, two 10-layer cell factories 3 are connected. The robotic arm connects the first liquid storage vehicle 2 with one of the 10-layer cell factories 3, so that the first liquid storage vehicle 2 can be connected to both 10-layer cell factories 3. The robotic arm operates to divide the cell suspension into two 10-layer cell factories 3, and the robotic arm assists in homogenizing the liquid (shaking lightly) so that the culture medium is evenly spread on the surface of each cell factory.

[0025] S230 , after separating the first liquid storage vehicle 2 from the 10-layer cell factory 3 with the assistance of a robotic arm, push the 10-layer cell factory 3 to a vehicle positioning system and then to a cell culture room for cultivation.

[0026] Further, combined with Figure 3 In step S300, the cells cultured in the 10-layer cell factory 3 are transferred to the 40-layer cell factory 5 for culture with the assistance of a robotic arm. The specific steps are: S310, placing each 10-layer cell factory 3 under a cell factory microscope to observe the culture conditions. After the culture conditions are met, the 10-layer cell factory 3 is moved to a liquid addition platform; Specifically, the cells cultured in each 10-layer cell factory 3 are observed. After the cells are observed and cultured to a desired state, the 10-layer cell factory 3 is moved to a liquid addition platform for adding digestion fluid.

[0027] S320, adding digestion fluid to the 10-layer cell factory 3 with the assistance of a robotic arm to complete digestion; S330, connecting the second liquid storage cart 4 to the 10-layer cell factory 3 via a robotic arm, so that the digested cells in the 10-layer cell factory 3 enter the second liquid storage cart 4; S340: After separating the second liquid storage cart 4 from the 10-layer cell factory 3 by a robotic arm, the second liquid storage cart 4 is connected to the 40-layer cell factory 5, and the digested cells in the 40-layer cell factory 5 are transported to each layer of the 40-layer cell factory 5; In some embodiments, for example, the number of the 40-layer cell factories 5 is not limited and can be 1, 2, 3, 4, or even more. Considering multiple 40-layer cell factories 5 as a 40-layer cell factory group, the second liquid storage vehicle 4 is connected to the 40-layer cell factory group. For example, when there are four 40-layer cell factories 5, the four 40-layer cell factories 5 are sequentially connected via pipes, and the second liquid storage vehicle 4 is connected to one of the 40-layer cell factories 5, thereby achieving communication between the second liquid storage vehicle 4 and the 40-layer cell factory group 5.

[0028] S350: After separating the second liquid storage vehicle 4 from the 40-layer cell factory 5 with the assistance of a robotic arm, the 40-layer cell factory 5 is pushed to a vehicle positioning system and then to a cell culture room for culture.

[0029] Furthermore, in step S320, digestion fluid is added to the 10-layer cell factory 3 with the assistance of a robotic arm, and the specific steps for completing digestion are as follows: pour out the old cell culture fluid, rotate the 10-layer cell factory 3 to the liquid adding position, add cell digestion fluid for the first time to wash the cell surface, discard the cell digestion fluid, and rotate the 10-layer cell factory 3 group to the liquid adding position; add cell digestion fluid to infiltrate the cell surface, place it on the cell factory group frame, and enter the cell digestion countdown; the robotic arm grabs the 10-layer cell factory 3, rotates 90° to observe the cell digestion situation, adds cell culture medium to terminate digestion, homogenizes the liquid, and oscillates it with a small amplitude and shakes it quickly with the assistance of the robotic arm; homogenizes the liquid again with the assistance of the robotic arm with a small amplitude and quick oscillation to complete digestion.

[0030] Further, combined with Figure 4 In step S400, the cells cultured in the 40-layer cell factory 5 are loaded into the third liquid storage vehicle 7 with the assistance of a robotic arm. The specific steps for use are as follows: S410, placing each 40-layer cell factory 5 under a cell factory microscope to observe the culture conditions. After the culture conditions are met, the 40-layer cell factory 5 is moved to a liquid addition platform; In some embodiments, illustratively, the cells cultured in each 40-layer cell factory 5 are observed. After the cells are observed and cultured to a desired state, the 40-layer cell factory 5 is moved to a liquid addition platform for adding digestion fluid.

[0031] S420, adding digestion fluid to the 40-layer cell factory 5 with the assistance of a robotic arm to complete digestion; S430 , connecting the third liquid storage cart 7 to the 40-layer cell factory 5 via a robotic arm, so that the digested cells in the 40-layer cell factory 5 enter the third liquid storage cart 7 .

[0032] Furthermore, in step S420, digestion fluid is added to the 40-layer cell factory 5 with the assistance of a robotic arm, and the specific steps for completing digestion are as follows: pour out the old cell culture fluid, rotate the 40-layer cell factory 5 to the liquid adding position, add cell digestion fluid for the first time to wash the cell surface, discard the cell digestion fluid, and rotate the 40-layer cell factory 5 group to the liquid adding position; add cell digestion fluid to infiltrate the cell surface, place it on the cell factory group frame, and enter the cell digestion countdown; the robotic arm grabs the 40-layer cell factory 5, rotates 90° to observe the cell digestion situation, adds cell culture medium to terminate digestion, homogenizes the liquid, and oscillates it with a small amplitude and shakes it quickly with the assistance of the robotic arm; homogenizes the liquid again with the assistance of a small amplitude and quick oscillation with the assistance of the robotic arm to complete the digestion.

[0033] Furthermore, the 2-layer cell factory 1, the 10-layer cell factory 3, and the 40-layer cell factory 5 all include a liquid inlet and a liquid outlet, wherein the liquid inlet is used to input cell fluid, and the liquid outlet is used to discharge cell fluid. The present invention separates the liquid inlet channel from the liquid outlet channel of each cell factory through the liquid inlet and the liquid outlet to avoid contamination of the cell fluid. In some embodiments, the liquid inlet pumps the cell digestion fluid, cell culture fluid, and cell suspension in the liquid storage vehicle into the cell factory group; the liquid outlet pumps the cell maintenance fluid, cell rinse fluid, and cell suspension in the cell factory group into each liquid storage vehicle 6 to complete the liquid exchange of the cell factory.

[0034] Combine Figure 5-8 The specific embodiments are given as follows Initial installation of CF2: CF2 (2-layer Cell Factory 1) includes ports A and B. Under a laminar flow hood, quickly insert the tubing's quick-connect plug into port B of CF2 (2-layer Cell Factory 1) and the capsule into port A. Use a pipetting machine to aseptically weld the right end of the tubing to the first reservoir cart 2. Once connected, open the tubing clamp, start the peristaltic pump, and slowly tilt CF2 until the cell suspension completely flows into the first reservoir cart 2. After transferring the cell suspension, turn off the peristaltic pump and disconnect the tubing from the first reservoir cart 2 using a tube sealer.

[0035] Installation and operation of the CF10 (10-layer cell factory 3) cell factory group: Under the laminar flow hood, pre-assemble the pipeline with CF10-1 and CF10-2 and push it to the cell factory group cart positioning system. Connect the pipeline to the first liquid storage cart 2 and use a pipe connection machine to complete the connection. Transfer the cell fluid from the first liquid storage cart 2 to CF10-1 and CF10-2. After a period of culture, start the automated operation program to ensure that CF10-1 and CF10-2 are connected to each liquid storage cart 6 and the second liquid storage cart 4. Execute the automated operation program to pump the old cell maintenance fluid and rinsing fluid of the CF10 cell factory group into the cell waste liquid cart. Next, use the cell digestion fluid cart to rinse and digest the CF10 cell factory group. Use the cell culture fluid cart to complete the neutralization treatment after digestion, prepare it into a cell suspension, and transfer it to the second liquid storage cart 4 to complete the digestion process of the CF10 cell factory group.

[0036] Installation and operation of the CF40 cell factory group: Under the laminar flow hood, connect the pipelines and capsule filters and install them to CF40-1, CF40-2, CF40-3 and CF40-4 (40-layer cell factory 5). Push it to the cell factory group cart positioning system, use the takeover machine to complete the connection between the pipelines, and set it aside. Run the automated operation program to connect the second liquid storage cart 4 with the CF40 cell factory group, and deliver the cell fluid to each CF40 cell factory for cell culture. Use the takeover machine to connect the pipeline to each liquid storage cart 6 and the third liquid storage cart 7 to ensure that the CF40 cell factory group is connected to each liquid storage cart 6 and the third liquid storage cart 7. Start the automated operation program and pump the old cell maintenance solution and rinsing solution of the CF40 cell factory group into the cell waste liquid cart. The cell digestion liquid cart is used to rinse and digest the CF40 cell factory group, and the cell culture liquid cart completes the neutralization treatment after digestion, prepares the treated liquid into a cell suspension, and distributes it to the third liquid storage cart 7 to complete the digestion process of the CF40 cell factory group.

[0037] As can be seen from the above embodiments, the present invention provides a new pipeline connection system for the operation of cell factories. The system can support the cultivation of multiple cell factories at the same time to form a cell factory group, and realize operations such as liquid addition and drainage through the pipeline system to complete cell inoculation, passage and cell suspension processing. At the same time, during the digestion process of processing one cell factory group, the system can flexibly intersperse the processing of other cell factory groups, thereby simplifying the operating procedures of multiple cell factories, significantly improving the working efficiency of cell culture, and effectively reducing the contamination risk of cell factories. In addition, the present invention overcomes the problems of large-scale cell culture in traditional cell factories, such as complex operation, large batch size, easy contamination, and uneven cell digestion.

[0038] Specifically, the piping system flexibly controls the connections between multiple cell factories. Using connectors such as reducers and straight-through pipes, it reliably connects wear-resistant tubing, silicone tubing, and thermoplastic tubing. It is compatible with cell factories of various sizes, such as CF10 and CF40. The system supports the flow of liquids into and out of any cell factory through the piping system, and pumping and draining operations are performed via a liquid addition platform, further improving operational efficiency.

[0039] In addition, the piping system is equipped with a cell factory adapter cap that includes butterfly filters and capsule filters to ensure a sterile environment within the cell factory and effectively balance the pressure inside and outside the cell factory. These designs not only ensure sterility during operation but also enhance the stability and safety of the system.

[0040] A specific embodiment is a cell culture piping system that uses silicone tubes, thermoplastic tubes, connectors, quick plugs, butterfly filters and capsule filters to place multiple cell factories in the same posture on the cell factory assembly rack to form a cell factory group. The cell factory has two liquid interfaces, one interface connects to the capsule filter to complete the circulation between the cell factory group and the outside air; the other interface connects the pipelines between the cell factories to complete the connection between the cell factories. The liquid inlet controls the peristaltic pump, robot and cell factory group operating table through the console, and can quantitatively pump the cell digestion solution, cell culture solution and cell suspension in the liquid storage vehicle into the cell factory group. The liquid outlet controls the peristaltic pump, robot and cell factory group operating table through the console, and can divert the cell maintenance solution, cell rinse solution and cell suspension in the cell factory group to each liquid storage vehicle 6 and the third liquid storage vehicle 7, completing the liquid discharge and exchange of the cell factory.

[0041] Specific embodiment, a cell culture pipeline system based on a robotic arm: The piping system for the 10-layer cell factory (CF10) assembly includes quick plugs, tube clamps, reducers, peristaltic pumps, straight-through tubes, wear-resistant tubes, thermoplastic tubes, and silicone tubes. The cell factory assembly and liquid storage vehicle pipelines are connected by a sterile pipe connection machine and a tube sealing machine.

[0042] The piping systems for digestion of the 10-layer cell factory group and packaging of the 40-layer cell factory group include quick plugs, tube clamps, straight-through tubes, wear-resistant tubes, thermoplastic tubes and silicone tubes. The cell factory group and liquid storage vehicle pipelines are connected by a sterile pipe connection machine and a tube sealing machine.

[0043] The piping systems of the 40-layer cell factory group's digestion and cell suspension 5L and 40L liquid storage carts include quick plugs, pipe clamps, straight-through pipes, wear-resistant pipes, thermoplastic pipes and silicone tubes. The cell factory group and liquid storage cart pipelines are connected by a sterile pipe connection machine and a tube sealing machine.

[0044] In a specific embodiment, the cell factory is positioned between a cell factory trolley positioning system, a cell factory operating table, a cell factory frame, and a cell factory microscope. The gripper and surveillance camera of a six-degree-of-freedom (6DOF) GMP-compliant robot are connected to the cell factory via quick-change clamps. The six-degree-of-freedom (6DOF) GMP-compliant robot is positioned in a fixed position within the room. All links are maintained in a GMP-compliant, sterile environment.

[0045] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0046] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cell culture method, characterized in that: The following steps are involved: S100, after the cells to be cultured are expanded and cultured, they are transferred to the second-layer cell factory for cell culture; S200, the cells cultured in the 2-layer cell factory are transferred to the 10-layer cell factory for culture with the assistance of a robotic arm; S300: The cells cultured in the 10-layer cell factory are transferred to the 40-layer cell factory for culture with the assistance of a robotic arm; S400: The cells cultured in the 40-layer cell factory are loaded into the third liquid storage vehicle with the assistance of a robotic arm for standby use.

2. The cell culture method according to claim 1, wherein In step S100 , the specific steps of expanding the cells to be cultured are: thawing and expanding the cells to be cultured in a T75 flask, and then digesting and transferring them into a two-layer cell factory for cell culture.

3. The cell culture method according to claim 1, wherein In step S200, the cells cultured in the 2-layer cell factory are transferred to the 10-layer cell factory for culture with the assistance of a robotic arm. The specific steps are: S210, digesting the cells cultured in the two-layer cell factory and then loading them into the first liquid storage vehicle; S220, connecting the first liquid storage vehicle to the 10-layer cell factory with the assistance of a robotic arm, so that the cell solution in the first liquid storage vehicle enters each layer of the 10-layer cell factory; S230: After separating the first liquid storage vehicle from the 10-layer cell factory with the assistance of a robotic arm, the 10-layer cell factory is pushed to a vehicle positioning system and then to a cell culture room for culture.

4. The cell culture method according to claim 3, wherein The number of the 10-layer cell factories is 2, and the two 10-layer cell factories are interconnected.

5. The cell culture method according to claim 4, characterized in that In step S300, the cells cultured in the 10-layer cell factory are transferred to the 40-layer cell factory for culture with the assistance of a robotic arm. The specific steps are: S310, placing each 10-layer cell factory under a cell factory microscope to observe the culture conditions. After the culture conditions are met, the 10-layer cell factory is moved to a liquid addition platform; S320, adding digestion fluid to the 10-layer cell factory with the assistance of a robotic arm to complete digestion; S330, connecting the second liquid storage vehicle to the 10-layer cell factory via a robotic arm, so that the digested cells in the 10-layer cell factory enter the second liquid storage vehicle; S340: After separating the second liquid storage vehicle from the 10-layer cell factory by a robotic arm, the second liquid storage vehicle is connected to the 40-layer cell factory, and the digested cells in the 40-layer cell factory are transported to each layer of the 40-layer cell factory; S350: After separating the second liquid storage vehicle from the 40-layer cell factory with the assistance of a robotic arm, the 40-layer cell factory is pushed to a vehicle positioning system and then to a cell culture room for culture.

6. The cell culture method according to claim 5, characterized in that In step S320, digestion fluid is added to the 10-layer cell factory with the assistance of a robotic arm. The specific steps for completing digestion are as follows: pour out the old cell culture fluid, rotate the 10-layer cell factory to the liquid adding position, add cell digestion fluid for the first time to wash the cell surface, discard the cell digestion fluid, and rotate the 10-layer cell factory group to the liquid adding position; add cell digestion fluid to infiltrate the cell surface, place it on the cell factory group frame, and enter the cell digestion countdown; the robotic arm grabs the 10-layer cell factory, rotates it 90° to observe the cell digestion situation, adds cell culture medium to terminate digestion, homogenizes the liquid, and oscillates it with a small amplitude and shakes it quickly with the assistance of the robotic arm; homogenizes the liquid again with the assistance of the robotic arm with a small amplitude and quick oscillation to complete digestion.

7. The cell culture method according to claim 1, wherein In step S400, the cells cultured in the 40-layer cell factory are loaded into the third liquid storage vehicle with the assistance of a robotic arm. The specific steps for use are as follows: S410, placing each 40-layer cell factory under a cell factory microscope to observe the culture conditions. After the culture conditions are met, the 40-layer cell factory is moved to a liquid addition platform; S420, adding digestion fluid to the 40-layer cell factory with the assistance of a robotic arm to complete digestion; S430, connecting the third liquid storage vehicle to the 40-layer cell factory via a robotic arm, so that the digested cells in the 40-layer cell factory enter the third liquid storage vehicle.

8. The cell culture method according to claim 7, characterized in that In step S420, digestion fluid is added to the 40-layer cell factory with the assistance of a robotic arm. The specific steps for completing digestion are as follows: pour out the old cell culture fluid, rotate the 40-layer cell factory to the liquid adding position, add cell digestion fluid for the first time to wash the cell surface, discard the cell digestion fluid, and rotate the 40-layer cell factory group to the liquid adding position; add cell digestion fluid to infiltrate the cell surface, place it on the cell factory group frame, and enter the cell digestion countdown; the robotic arm grabs the 40-layer cell factory, rotates 90° to observe the cell digestion situation, adds cell culture medium to terminate digestion, homogenizes the liquid, and oscillates it with a small amplitude and shakes it quickly with the assistance of the robotic arm; homogenizes the liquid again with the assistance of the robotic arm with a small amplitude and quick oscillation to complete the digestion.

9. The cell culture method according to claim 1, wherein The number of the 40-layer cell factories is 4, and the 4 40-layer cell factories are interconnected.

10. The cell culture method according to claim 1, characterized in that The 2-layer cell factory, the 10-layer cell factory and the 40-layer cell factory all include a liquid inlet and a liquid outlet. The liquid inlet is used to input liquid, and the liquid outlet is used to discharge liquid.

Images