Closed magnetic bead incubation method, equipment, medium and program product

Abstract

The invention provides a closed magnetic bead incubation method, equipment, a medium and a program product, and relates to the technical field of biology and medical treatment. The method comprises the following steps: controlling a magnetic bead incubation structure in which magnetic beads are pre-encapsulated to be placed into a magnetic frame, fixing the magnetic bead incubation structure, and standing the magnetic bead incubation structure for a first preset time; the pipeline change-over switch is controlled to switch to open the waste liquid outflow pipeline; pBS or a culture medium is injected into the incubation part through the liquid injection part, and the magnetic bead preservation liquid in the magnetic bead incubation structure is replaced through pressure; a pipeline change-over switch is controlled to be switched to open a cell bag connecting pipeline, and the magnetic bead incubation structure is controlled to be moved out of the magnetic frame to resuspend the magnetic beads; the magnetic bead suspension is injected into the cell bag filled with cells through compressed air of the liquid injection part, and the cell bag is connected with the cell bag connecting pipeline. By arranging the matched magnetic bead incubation structure and the magnetic frame, a totally-enclosed magnetic bead incubation method is developed, and the advantages of reducing pollution risks, improving operation efficiency and space utilization, achieving modular management and the like are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biology and medical technology, and more specifically, to a closed magnetic bead incubation method, equipment, medium and program product. Background Art

[0002] At present, the micron-scale CD3 / CD28 sorting activation magnetic beads are packed in vials, which need to be punctured with a syringe to absorb the magnetic beads. The risks of syringe puncturing vials mainly include the following: (1) Increased contamination risk: Traditional vial packaging materials may increase the risk of bacterial contamination during the puncture process. (2) Puncture debris, causing adverse reactions to infusion: When the syringe needle punctures the vial stopper, debris may be generated. Once these debris enter the cell drug, it will seriously threaten the life safety of the patient taking the drug. (3) Needle stick risk: When using a syringe barrel to puncture the vial, it is easy to bring the risk of needle stick injury. (4) Drug solution residue: Pharmaceutical companies usually fill an excess volume of drug solution to compensate for the loss of drug solution caused by drug aspiration or transfer using a needle and syringe. (5) High environmental requirements: Traditional vial packaging materials must be punctured by syringe barrels in a Class A environment to remove magnetic beads, which requires high environmental cleanliness. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a closed magnetic bead incubation method, device, medium and program product; the method of the present invention develops a fully closed magnetic bead incubation method by setting a matching magnetic bead incubation structure and a magnetic frame, so as to achieve the advantages of reducing contamination risk, improving operation efficiency and space utilization, modular management, etc.

[0004] The first aspect of the present application discloses a closed magnetic bead incubation method, the method comprising:

[0005] S101, control the magnetic bead incubation structure with pre-filled magnetic beads inside to be placed in a magnetic rack and fixed, and let it stand for a first predetermined time; the magnetic bead incubation structure includes a liquid injection part, an incubation part and a liquid outflow part connected in sequence from top to bottom; the liquid outflow part includes a cell bag connecting pipeline and a waste liquid outflow pipeline connected to the incubation part, and a pipeline conversion switch for controlling whether the cell bag connecting pipeline and the waste liquid outflow pipeline are connected;

[0006] S102, control the pipeline conversion switch to switch the waste liquid outflow pipeline to open;

[0007] S103, injecting a first predetermined amount of PBS or culture medium into the incubation part through the liquid injection part, and replacing the magnetic bead preservation liquid inside the magnetic bead incubation structure by pressure;

[0008] S104, control the pipeline conversion switch to switch to the cell bag connection pipeline to open, control the magnetic bead incubation structure to be moved out of the magnetic rack, and resuspend the magnetic beads;

[0009] S105, injecting the magnetic bead suspension into the cell bag containing the cells through the compressed air of the liquid injection part, and connecting the cell bag to the cell bag connecting pipeline.

[0010] In some embodiments, the method further includes S106, aspirating the culture medium containing the cells into the incubation part through the liquid injection part to reduce the residual magnetic beads.

[0011] The second aspect of the present application discloses a fully enclosed cell production method, the method comprising:

[0012] S201, obtaining a peripheral blood sample, and isolating PBMC from the peripheral blood sample;

[0013] S202, incubating the PBMC with magnetic beads using the method described in the first aspect of the present application to screen and obtain target cells;

[0014] S203, sorting the target cells using magnetic bead sorting technology to obtain sorted cells;

[0015] S204, culturing the sorted cells;

[0016] Optionally, magnetic beads are pre-filled inside the magnetic bead incubation structure.

[0017] The third aspect of the present application discloses a computer device, which includes: a memory and a processor; the memory is used to store a computer program; and the processor executes the computer program to implement the steps of the above method.

[0018] A fourth aspect of the present application discloses a computer-readable storage medium having a computer program stored thereon, wherein the computer program implements the steps of the above method when executed by a processor.

[0019] A fifth aspect of the present application discloses a computer program product, including a computer program, which implements the steps of the above method when executed by a processor.

[0020] The sixth aspect of the present application discloses a closed magnetic bead incubation device, which is used for the magnetic bead incubation method disclosed in the first aspect of the present application, and for the fully closed cell production method disclosed in the second aspect of the present application, and the device comprises: a magnetic bead incubation structure and a magnetic frame;

[0021] The magnetic bead incubation structure comprises a liquid injection part, an incubation part and a liquid outflow part which are sequentially connected from top to bottom; the liquid outflow part comprises a cell bag connection pipeline and a waste liquid outflow pipeline which are connected to the incubation part, and a pipeline conversion switch for controlling whether the cell bag connection pipeline and the waste liquid outflow pipeline are connected;

[0022] The magnetic frame is provided with a limiting hole for placing the liquid outflow part.

[0023] In some embodiments, the liquid injection portion is configured as an inflow conduit, and the inflow conduit is provided with an opening for connecting a syringe;

[0024] Optionally, the incubation part is a sealed structure;

[0025] Optionally, the waste liquid outflow pipeline is arranged on one side of the cell bag connecting pipeline, and a pipeline conversion switch is arranged at the connection between the cell bag connecting pipeline and the waste liquid outflow pipeline, or pipeline conversion switches are respectively arranged on the cell bag connecting pipeline and the waste liquid outflow pipeline;

[0026] Optionally, the cell bag connecting pipeline and the waste liquid outflow pipeline are independently connected to the incubation part, and switches are respectively provided on the cell bag connecting pipeline and the waste liquid outflow pipeline.

[0027] In some embodiments, a first filter membrane is disposed in the inflow conduit;

[0028] Optionally, a second filter membrane is arranged in the waste liquid outflow pipeline.

[0029] In some embodiments, the magnetic frame includes a magnetic frame body and a magnetic fixing member, a placement space is provided on one side of the magnetic frame body, and an end of the placement space away from the magnetic frame body is an opening; the magnetic fixing member has two ends, and the two ends of the magnetic fixing member are respectively provided on the left and right sides of the opening of the placement space;

[0030] Optionally, a bottom bracket is provided at the bottom of the placement space, and the bottom bracket is connected to the main body of the magnetic frame;

[0031] Optionally, the limiting hole is arranged on the bottom bracket, and a side of the limiting hole away from the magnetic frame body is arranged as an opening.

[0032] This application has the following beneficial effects:

[0033] Fully closed cell production systems have many advantages in the field of cell therapy. Here are some key points:

[0034] 1. Reduce contamination risk: The fully enclosed system greatly reduces the risk of contamination by reducing manual contact points. In the traditional cell preparation process, the operation of opening and closing the container lid multiple times will bring a high risk of contamination. The fully enclosed system integrates multiple steps into one device, reducing the number of processing steps and the contact between the sterile boundary, thereby reducing the risk of contamination.

[0035] 2. Improve operational efficiency and space utilization: The fully enclosed system integrates multiple devices into the same device, which is small in size. It can reduce the floor space while ensuring low contamination risk, make more effective use of the clean room area and save costs.

[0036] 3. Modular management, customized by product: The fully closed system can manage different steps in a modular way and modify modules at any time according to production needs, so as to customize the workflow and platform selection that best suits the customer's products according to their needs.

[0037] 4. Reduce labor costs: Fully automated closed cell therapy processing systems are usually equipped with intuitive program software for user operation, design and management. Because there is no need for operators to directly handle cells, the training costs for employee operations are reduced, making the production process more convenient.

[0038] 5. Improve product reliability and repeatability: The fully automated control system reduces human errors and improves product reliability and repeatability.

[0039] 6. Functional expansion and flexibility: Fully enclosed systems such as Lonza's Cocoon® platform are suitable for the production of suspended cells and adherent cells. They integrate multiple steps such as magnetic bead sorting, viral transduction or cell transfection, cell activation, proliferation, and harvesting, simplifying the upstream and downstream cell processing processes from formulation to final product, forming a complete cell solution.

[0040] 7. Fully enclosed system improves safety: Cocoon® fully enclosed disposable cassette consumables provide dual-zone control at 37°C and 4°C. By reducing manual contact points, it reduces the risk of human error and environmental cleanliness, and improves the overall safety of the product.

[0041] 8. Easy implementation of industrialization: The fully enclosed system enhances controllability and replicability, can effectively meet the product's transition from PD production process to IND, and complies with GMP production requirements. It only needs to be scaled up in parallel to quickly implement industrialization.

[0042] These advantages make the fully closed cell production system increasingly important in the field of cell therapy, and help achieve safer, more efficient and scalable production of cell therapy products. At present, there are commercially available products of bagged culture medium and PBS, which can be directly combined with the fully closed cell production system, but there are no corresponding closed packaging products for cytokines and magnetic beads related to cell culture. Therefore, the present invention designs a closed packaging material for magnetic beads to make the fully closed cell production system more perfect. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0044] Figure 1 Schematic diagram of the closed magnetic bead incubation method provided by the first aspect of the embodiment of the present invention;

[0045] Figure 2 is a schematic diagram of a fully enclosed cell production method provided by the second aspect of an embodiment of the present invention;

[0046] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention;

[0047] Figure 4 is a schematic diagram of the architecture of an exemplary computing device provided by an embodiment of the present invention;

[0048] Figure 5 is a schematic diagram of a storage medium provided by an embodiment of the present invention;

[0049] Figure 6 is a schematic diagram of a closed magnetic bead incubation system provided in an embodiment of the present invention;

[0050] Figure 7 is a schematic diagram of a fully enclosed cell production system provided by an embodiment of the present invention;

[0051] Figure 8 It is a schematic diagram of the structure of magnetic beads incubation provided by an embodiment of the present invention;

[0052] Fig. 9 It is a schematic diagram of a magnetic bead incubation structure in which a cell bag connection pipeline and a waste liquid outflow pipeline are independently connected to an incubation part provided in an embodiment of the present invention;

[0053] Fig.10 is a schematic diagram of the structure of a magnetic frame provided by an embodiment of the present invention;

[0054] In the figure, 1. Magnetic frame body; 2. Magnetic fixing part; 3. Placement space; 4. Bottom support; 5. Limiting hole; 6. Inflow pipeline; 7. Cell bag connecting pipeline; 8. Waste liquid outflow pipeline; 9. Pipeline conversion switch; 10. Second filter membrane; 11. First filter membrane; 12. Filling port; 13. Incubation part. DETAILED DESCRIPTION

[0055] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0056] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0058] Figure 1 Schematic diagram of a closed magnetic bead incubation method provided by an embodiment of the present invention, specifically, the method comprises the following steps: S101, controlling a magnetic bead incubation structure with internal pre-filled magnetic beads to be placed in a magnetic rack and fixed, and standing for a first predetermined time; the magnetic bead incubation structure comprises a liquid injection part, an incubation part and a liquid outflow part which are sequentially connected from top to bottom; the liquid outflow part comprises a cell bag connecting pipeline and a waste liquid outflow pipeline which are connected to the incubation part, and a pipeline conversion switch for controlling whether the cell bag connecting pipeline and the waste liquid outflow pipeline are connected;

[0059] S102, control the pipeline conversion switch to switch the waste liquid outflow pipeline to open;

[0060] S103, injecting a first predetermined amount of PBS or culture medium into the incubation part through the liquid injection part, and replacing the magnetic bead preservation liquid inside the magnetic bead incubation structure by pressure;

[0061] S104, control the pipeline conversion switch to switch to the cell bag connection pipeline to open, control the magnetic bead incubation structure to be moved out of the magnetic rack, and resuspend the magnetic beads;

[0062] S105, injecting the magnetic bead suspension into the cell bag containing the cells through the compressed air of the liquid injection part, and connecting the cell bag to the cell bag connecting pipeline.

[0063] In some embodiments, if there are residual magnetic beads in S105, the method further includes S106, aspirating the culture medium containing the cells into the incubation part through the liquid injection part to reduce the residual magnetic beads.

[0064] In some embodiments, the present application also discloses a closed magnetic bead incubation system, such as Figure 6 As shown, the system comprises:

[0065] The magnetic bead incubation structure and magnetic rack combination module 301 is used or configured to control the magnetic bead incubation structure with internal pre-filled magnetic beads to be placed in the magnetic rack and fixed, and left to stand for a first predetermined time; the magnetic bead incubation structure includes a liquid injection part, an incubation part and a liquid outflow part connected in sequence from top to bottom; the liquid outflow part includes a cell bag connection pipeline and a waste liquid outflow pipeline connected to the incubation part, and a pipeline conversion switch for controlling whether the cell bag connection pipeline and the waste liquid outflow pipeline are connected;

[0066] The first pipeline control module 302 is used or configured to control the pipeline conversion switch to switch to open the waste liquid outflow pipeline;

[0067] The preservation liquid replacement module 303 is used or configured to inject a first predetermined amount of PBS or culture medium into the incubation part through the liquid injection part, and replace the magnetic bead preservation liquid inside the magnetic bead incubation structure by pressure;

[0068] The second pipeline control module 304 is used or configured to control the pipeline conversion switch to switch to the cell bag connection pipeline to open, control the magnetic bead incubation structure to be moved out of the magnetic frame, and resuspend the magnetic beads;

[0069] The complex binding module 305 is used for or configured to inject the magnetic bead suspension into the cell bag containing cells through the compressed air of the liquid injection part, and the cell bag is connected to the cell bag connecting pipeline.

[0070] In some embodiments, the closed magnetic bead incubation system further includes: a magnetic bead residue cleaning module, which is used for or configured to pump the culture medium containing cells into the incubation part through the liquid injection part to reduce the residue of magnetic beads.

[0071] In some embodiments, the second aspect of the present application discloses a fully enclosed cell production method, such as Figure 2 As shown, the method includes:

[0072] S201, obtaining a peripheral blood sample, and isolating PBMC from the peripheral blood sample;

[0073] S202, incubating the PBMC with magnetic beads using the method described in the first aspect of the present application to screen and obtain target cells;

[0074] S203, sorting the target cells using magnetic bead sorting technology to obtain sorted cells;

[0075] S204, culturing the sorted cells; optionally, the magnetic beads are pre-filled in the magnetic bead incubation structure.

[0076] In some embodiments, the present application also discloses a fully enclosed cell production system, such as Figure 7 As shown, the system comprises:

[0077] The PBMC separation module 401 is used or configured to obtain a peripheral blood sample and separate PBMC from the peripheral blood sample; the separation method includes density gradient centrifugation;

[0078] A magnetic bead incubation module 402, used for or configured to perform magnetic bead incubation on the PBMCs using the method described in the first aspect of the present application to screen and obtain target cells;

[0079] A cell sorting module 403 is used for or configured to sort the target cells using magnetic bead sorting technology to obtain sorted cells;

[0080] The cell culture module 404 is used for or configured to culture the sorted cells.

[0081] In some embodiments, the sixth aspect of the present application discloses a closed magnetic bead incubation device, which is used for the magnetic bead incubation method disclosed in the first aspect of the present application, and for the fully closed cell production method disclosed in the second aspect of the present application, and the device includes: a magnetic bead incubation structure and a magnetic frame;

[0082] like Figure 8 and Fig. 9 As shown, the magnetic bead incubation structure includes a liquid injection part, an incubation part 13 and a liquid outflow part which are sequentially connected from top to bottom; the liquid outflow part includes a cell bag connection pipeline 7 and a waste liquid outflow pipeline 8 which are connected to the incubation part 13, and a pipeline conversion switch 9 for controlling whether the cell bag connection pipeline 7 and the waste liquid outflow pipeline 8 are connected;

[0083] In some embodiments, the liquid injection part is configured as an inflow pipe 6, and the inflow pipe 6 is provided with an opening for connecting a syringe; in some embodiments, a first filter membrane 11 is provided in the inflow pipe 6, and the first filter membrane 11 plays a blocking and air permeability role;

[0084] In some more specific embodiments, the incubation portion 13 is a sealed structure; in some specific embodiments, the outer shape of the incubation portion 13 is an arc shape.

[0085] In some embodiments, the waste liquid outflow pipeline 8 is arranged on one side of the cell bag connection pipeline 7, and a pipeline conversion switch 9 is arranged at the connection between the cell bag connection pipeline 7 and the waste liquid outflow pipeline 8, or a pipeline conversion switch 9 is respectively arranged on the cell bag connection pipeline 7 and the waste liquid outflow pipeline 8; Optionally, the cell bag connection pipeline 7 and the waste liquid outflow pipeline 8 are independently connected to the incubation part 13, and switches are respectively arranged on the cell bag connection pipeline 7 and the waste liquid outflow pipeline 8. Optionally, a second filter membrane 10 is arranged in the waste liquid outflow pipeline 8.

[0086] In another specific embodiment, when the pipeline conversion switch 9 is set at the connection between the cell bag connecting pipeline 7 and the waste liquid outflow pipeline 8, the pipeline conversion switch 9 is set as a three-way valve; when the pipeline conversion switch 9 is respectively set in the cell bag connecting pipeline 7 and the waste liquid outflow pipeline 8, the pipeline conversion switch 9 is set as a clamp or other switches that are convenient for controlling the flow.

[0087] In one embodiment, the cell bag connecting pipeline 7 and the waste liquid outflow pipeline 8 are independently connected to the incubation part 13, and switches are respectively set on the cell bag connecting pipeline 7 and the waste liquid outflow pipeline 8. In a specific implementation scheme, the switches on the two pipelines are respectively set as clamps, which play the role of controlling the flow or cutoff of liquid in different pipelines.

[0088] In some embodiments, a filling port 12 is provided in the inflow pipe 6 between the first filter membrane 11 and the incubation part 13, and heat-sealed after filling; or, a filling port 12 is provided on one side of the incubation part 13. The filling port 12 is used as a filling port for the preparation magnetic beads, and is sealed after filling. Figure 8 and Fig. 9 As shown, the filling port 12 is arranged on one side of the incubation part 13 .

[0089] In some embodiments, the inflow pipeline 6 is provided with an opening, the opening is connected to a syringe, and liquid is pushed into the incubation part 13 through the syringe.

[0090] In some embodiments, the pore size of the first filter membrane 11 or the second filter membrane 10 is 0.22 μm.

[0091] In some embodiments, the cell bag connection line 7 is connected to a peristaltic pump.

[0092] In some embodiments, the waste liquid outflow pipe 8 is connected to other bags for containing waste liquid, in which case a second filter membrane 10 is required; if it is directly a waste liquid bag, the second filter membrane 10 is not required.

[0093] In some embodiments, Fig.10 As shown, the magnetic frame includes a magnetic frame body 1 and a magnetic fixing part 2. A placement space 3 is arranged on one side of the magnetic frame body 1, and the end of the placement space 3 away from the magnetic frame body 1 is an opening; the magnetic fixing part 2 has two ends, and the two ends of the magnetic fixing part 2 are respectively arranged on the left and right sides of the opening of the placement space 3; the magnetic frame is provided with a limiting hole 5 for placing the liquid outflow part.

[0094] In some embodiments, one of the two ends of the magnetic fixing member 2 can be separated relative to the left or right side of the opening of the placement space 3.

[0095] In some more specific embodiments, the two ends of the magnetic fixing member 2 are respectively a first end and a second end, the first end or the second end is fixedly arranged on the left side of the opening of the placement space 3 or can rotate relative to the left side of the opening of the placement space 3, and the second end or the first end can be separated relative to the right side of the opening of the placement space 3.

[0096] In some embodiments, a bottom support 4 is provided at the bottom of the placement space 3, and the bottom support 4 is connected to the magnetic frame body 1;

[0097] In some embodiments, the limiting hole 5 is arranged on the bottom bracket 4, and the side of the limiting hole 5 away from the magnetic frame body 1 is arranged to be an opening;

[0098] In some embodiments, at least one limiting hole 5 is provided; the number of limiting holes 5 is set to be arc-shaped or square-shaped. The number of limiting holes 5 is determined according to the setting mode and number of the cell bag connecting pipeline 7 and the waste liquid outflow pipeline 8. Fig.10 As shown, one limiting hole 5 is provided and is arranged in an arc shape.

[0099] In some embodiments, at least one magnetic fixing member 2 is provided. In some more specific embodiments, the magnetic fixing member 2 is provided as a fixing belt. Figure 1 and Figure 2 As shown, the first end of the left side of the magnetic fixing belt is fixed or can rotate relative to the opening of the placement space 3; the right side of the magnetic fixing belt can be separated relative to the opening of the placement space 3. Fig.10 As shown, one magnetic fixing member 2 is provided.

[0100] In a specific embodiment, the magnetic frame is a rectangular parallelepiped, and in the top view or bottom view of the magnetic frame, the placement space 3 is arc-shaped.

[0101] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention, such as Figure 3 As shown, the device 2000 may include: one or more processors 2010, and one or more memories 2020; wherein the memories store computer-readable codes, and when the computer-readable codes are run by the one or more processors, the method described above may be executed.

[0102] The processor in this embodiment can be an integrated circuit chip with signal processing capabilities. The above processor can be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components. The disclosed methods, operations and logic block diagrams in the embodiments of the present disclosure can be implemented or executed. The general-purpose processor can be a microprocessor or the processor can also be any conventional processor, etc., which can be an X86 architecture or an ARM architecture.

[0103] In general, various example embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the disclosed embodiments are illustrated or described as block diagrams, flow charts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented in hardware, software, firmware, dedicated circuits or logic, general purpose hardware or controllers or other computing devices, or some combination thereof as non-limiting examples.

[0104] For example, the method or device according to the embodiment of the present disclosure may also be implemented by Figure 4 The architecture of the computing device 3000 shown in FIG. Figure 4 As shown, the computing device 3000 may include a bus 3010, one or more CPUs 3020, a read-only memory (ROM) 3030, a random access memory (RAM) 3040, a communication port 3050 connected to a network, an input / output component 3060, a hard disk 3070, etc. The storage device in the computing device 3000, such as ROM 3030 or hard disk 3070, may store various data or files used for processing and / or communication of the method provided by the present disclosure and program instructions executed by the CPU. The computing device 3000 may also include a user interface 3080. Of course, Figure 4 The architecture shown is only exemplary and can be omitted according to actual needs when implementing different devices. Figure 4 One or more components of a computing device are shown.

[0105] The embodiment of the present invention also provides a computer-readable storage medium, such as Figure 5 As shown, it is a schematic diagram of a storage medium 4000 provided in an embodiment of the present invention, and a computer readable instruction 4010 is stored on the computer storage medium 4020. When the computer readable instruction 4010 is executed by a processor, the method according to the embodiment of the present disclosure described with reference to the above figures can be executed. The computer readable storage medium in the embodiment of the present disclosure can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. The non-volatile memory can be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM) or a flash memory. The volatile memory can be a random access memory (RAM), which is used as an external cache. By way of example and not limitation, many forms of RAM are available, such as static random access memory (SRAM), dynamic random access memory (DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDRSDRAM), enhanced synchronous dynamic random access memory (ESDRAM), synchronous link dynamic random access memory (SLDRAM), and direct memory bus random access memory (DR RAM). It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory. It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory.

[0106] The embodiments of the present disclosure further provide a computer program product or system, including a computer program, which implements the steps of the above method when executed by a processor. Specific embodiments

[0107] A fully enclosed instrument with PBMC (peripheral blood mononuclear cell) separation, magnetic bead incubation, cell sorting and cell culture functions, usually used for automated cell sorting and processing, and widely used in immunology research, cell therapy, clinical diagnosis and biomedical research. The working principle and process of this type of instrument are as follows:

[0108] 1. PBMC Isolation:

[0109] PBMCs (peripheral blood mononuclear cells) are usually isolated from peripheral blood samples. A common isolation method is density gradient centrifugation (such as Ficoll-Paque), which separates cells based on their density differences.

[0110] Working principle: Through centrifugal force, various types of cells in the blood (red blood cells, white blood cells, platelets, etc.) are layered according to their density. PBMCs are distributed in the density gradient layer of Ficoll liquid. The instrument is usually equipped with a centrifugal system or uses other separation technologies (such as microfluidic chips) to achieve PBMC separation. The separated PBMCs are subsequently incubated with magnetic beads.

[0111] 2. Magnetic bead incubation:

[0112] Magnetic bead incubation is to selectively enrich specific types of cells by combining magnetic particles with specific antibodies on the cell surface. For example, magnetic beads with anti-CD3, CD4, CD8 and other antibodies are used to enrich T cells, or anti-CD19 magnetic beads are used to sort B cells.

[0113] Working principle: The surface of magnetic beads binds to marker molecules on the cell surface (such as CD molecules) to form a magnetic bead-cell complex. Then, through the action of an external magnetic field, the magnetic bead-cell complex is adsorbed to a specific area, thereby separating the cell population of interest. The magnetic beads are usually brought into contact with the cells through liquid flow or rotation to complete the incubation process. There is currently no fully enclosed consumables for this process. The design of this enclosed packaging material is used to optimize the magnetic bead addition process in this step.

[0114] 3. Cell sorting:

[0115] Magnetic bead sorting technology (MACS) is used to sort cells bound to magnetic beads. Cells are selected or eliminated through the magnetic field to achieve the purpose of sorting.

[0116] 4. Cell culture:

[0117] The sorted cells need to be cultured in an environment suitable for their growth. The cell culture system usually includes environmental control functions such as temperature control, humidity control, and carbon dioxide concentration regulation.

[0118] Working principle: In a fully enclosed instrument, the culture system usually uses an automated culture box or culture dish that can accurately control the temperature (usually 37°C), CO 2 Concentration (usually 5%), humidity, etc. Cells will obtain the required nutrients in a specific culture medium to grow, divide and maintain life activities. Automated equipment may also include the function of adding drugs or nutrients to support the continuous culture of cells.

[0119] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the possible architecture, functions and operations of the systems, methods and computer program products according to various embodiments of the present disclosure. In this regard, each box in the flowchart or block diagram can represent a module, a program segment, or a part of a code, and the module, program segment, or a part of the code contains one or more executable instructions for implementing the specified logical function. It should also be noted that in some alternative implementations, the functions marked in the box can also occur in a different order from the order marked in the accompanying drawings. For example, two boxes represented in succession can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each box in the block diagram and / or flowchart, and the combination of boxes in the block diagram and / or flowchart can be implemented with a dedicated hardware-based system that performs a specified function or operation, or can be implemented with a combination of dedicated hardware and computer instructions.

[0120] In general, various example embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the disclosed embodiments are illustrated or described as block diagrams, flow charts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented in hardware, software, firmware, dedicated circuits or logic, general purpose hardware or controllers or other computing devices, or some combination thereof as non-limiting examples.

[0121] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0122] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0123] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0124] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0125] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. It should be understood by those skilled in the art that various modifications and combinations may be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A closed magnetic bead incubation method, characterized in that: The method comprises: S101, controlling the magnetic bead incubation structure to be placed in a magnetic rack for fixation and to stand for a first predetermined time; the magnetic bead incubation structure comprises a liquid injection part, an incubation part and a liquid outflow part which are sequentially connected from top to bottom; the liquid outflow part comprises a cell bag connection pipeline and a waste liquid outflow pipeline which are connected to the incubation part, and a pipeline conversion switch for controlling whether the pipeline is connected; S102, control the pipeline conversion switch to switch the waste liquid outflow pipeline to open; S103, injecting a first predetermined amount of PBS or culture medium into the incubation part through the liquid injection part, and replacing the magnetic bead preservation liquid inside the magnetic bead incubation structure by pressure; S104, control the pipeline conversion switch to switch to the cell bag connection pipeline to open, control the magnetic bead incubation structure to be moved out of the magnetic rack, and resuspend the magnetic beads; S105, injecting the magnetic bead suspension into the cell bag through compressed air from the liquid injection part, and connecting the cell bag to the cell bag connecting pipeline.

2. The closed magnetic bead incubation method according to claim 1, characterized in that: The method further includes S106, aspirating the culture medium containing the cells to the incubation part through the liquid injection part to reduce the residual magnetic beads.

3. A fully enclosed cell production method, characterized in that: The method comprises: S201, obtaining a peripheral blood sample, and isolating PBMC from the peripheral blood sample; S202, incubating the PBMC with magnetic beads using the method described in any one of claims 1 to 2 to screen and obtain target cells; S203, sorting the target cells using magnetic bead sorting technology to obtain sorted cells; S204, culturing the sorted cells; Optionally, magnetic beads are pre-filled inside the magnetic bead incubation structure.

4. A computer device, characterized in that: The device comprises: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the method according to any one of claims 1 to 3.

5. A computer-readable storage medium, characterized in that: A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to any one of claims 1 to 3 are implemented.

6. A computer program product comprising a computer program, characterized in that When the computer program is executed by a processor, the steps of the method described in any one of claims 1 to 3 are implemented.

7. A closed magnetic bead incubation device, the closed magnetic bead incubation device is used for the magnetic bead incubation method according to any one of claims 1 to 2, and for the fully closed cell production method according to claim 3, characterized in that: The device comprises: a magnetic bead incubation structure and a magnetic frame; The magnetic bead incubation structure comprises a liquid injection part, an incubation part and a liquid outflow part which are sequentially connected from top to bottom; the liquid outflow part comprises a cell bag connection pipeline and a waste liquid outflow pipeline which are connected to the incubation part, and a pipeline conversion switch for controlling whether the cell bag connection pipeline and the waste liquid outflow pipeline are connected; The magnetic frame is provided with a limiting hole for placing the liquid outflow part.

8. The closed magnetic bead incubation device according to claim 7, characterized in that: The liquid injection part is configured as an inflow pipeline, and the inflow pipeline is provided with an opening for connecting a syringe; Optionally, the incubation part is a sealed structure; Optionally, the waste liquid outflow pipeline is arranged on one side of the cell bag connecting pipeline, and a pipeline conversion switch is arranged at the connection between the cell bag connecting pipeline and the waste liquid outflow pipeline, or pipeline conversion switches are respectively arranged on the cell bag connecting pipeline and the waste liquid outflow pipeline; Optionally, the cell bag connecting pipeline and the waste liquid outflow pipeline are independently connected to the incubation part, and switches are respectively provided on the cell bag connecting pipeline and the waste liquid outflow pipeline.

9. The closed magnetic bead incubation device according to claim 7, characterized in that: Disposing a first filter membrane in the inflow pipeline; Optionally, a second filter membrane is arranged in the waste liquid outflow pipeline.

10. The closed magnetic bead incubation device according to claim 7, characterized in that: The magnetic frame comprises a magnetic frame body and a magnetic fixing part, a placement space is arranged on one side of the magnetic frame body, and an end of the placement space away from the magnetic frame body is an opening; the magnetic fixing part has two ends, and the two ends of the magnetic fixing part are respectively arranged on the left and right sides of the opening of the placement space; Optionally, a bottom bracket is provided at the bottom of the placement space, and the bottom bracket is connected to the main body of the magnetic frame; Optionally, the limiting hole is arranged on the bottom bracket, and a side of the limiting hole away from the main body of the magnetic frame is arranged as an opening; Optionally, at least one limiting hole is provided; Optionally, at least one magnetic fixing member is provided.

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