A method for rapidly preparing CAR-T cells by using a full-automatic cell preparation instrument

Abstract

The application discloses a kind of methods for rapidly preparing CAR-T cells using full-automatic cell preparation instrument, it is related to CAR-T cell preparation technical field, comprising the following steps: step 1, peripheral blood mononuclear cell separation;Step 2, T cell activation and CAR vector transduction;Step 3, CAR-T cell expansion;Step 4, washing and concentrating;Step 5, quality detection.The method for rapidly preparing CAR-T cells using full-automatic cell preparation instrument, relies on full-automatic cell preparation instrument to complete whole-process closed automatic preparation, can greatly shorten the preparation period of CAR-T cell, improve the timeliness of clinical treatment, without manual operation throughout can effectively reduce the risk of cell contamination, while eliminating the product fluctuation caused by artificial and environmental difference, guarantee the standardization and consistency of cell preparation, the cell activity prepared is excellent, can significantly improve the clinical application value and popularity of CAR-T therapy.

Description

Technical Field

[0001] This invention relates to the field of CAR-T cell preparation technology, specifically a method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument. Background Technology

[0002] CAR-T cell therapy has made significant breakthroughs in the treatment of hematological malignancies such as leukemia and lymphoma. However, traditional preparation processes have many limitations. Traditional methods usually need to be carried out in large GMP workshops, rely on manual operation, and have a complicated process. From cell collection to reinfusion, it often takes 10-14 days. The long cycle not only increases costs, but may also cause some patients to miss the best treatment opportunity due to disease progression.

[0003] In addition, traditional preparation processes require multiple cell transfers, which carries a high risk of contamination, and differences between different operators and laboratories may affect product consistency. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument, thus solving the problems mentioned in the background section.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument, comprising the following steps:

[0006] Step 1: Isolation of peripheral blood mononuclear cells:

[0007] The patient's peripheral blood sample was added to the separation module of the fully automated cell preparation instrument, and PBMCs were obtained by density gradient centrifugation.

[0008] Step 2, T cell activation and CAR vector transduction:

[0009] PBMCs were transferred into a reaction module, and activating reagents and viral vectors carrying CAR sequences were added for simultaneous activation and transduction.

[0010] Step 3, CAR-T cell expansion:

[0011] In the culture module, cells are cultured for 1-2 days under preset temperature, gas concentration, and cytokine conditions;

[0012] Step 4, Washing and Concentrating:

[0013] The magnetic beads, free viruses, and metabolites were removed by the purification module, and CAR-T cells were obtained by concentration.

[0014] Step 5, Quality Inspection:

[0015] Detect cell viability, CAR positivity rate, and sterility indicators;

[0016] Steps 1 through 5 are all completed in a fully automated cell preparation instrument, with a total preparation cycle of 2-3 days.

[0017] Furthermore, in step 2, the activating agent is anti-CD3 / CD28 magnetic beads, and the ratio of magnetic beads to cells is 1:1 to 2:1.

[0018] Furthermore, in step 2, the viral vector is a lentivirus or a retrovirus with an MOI value of 2-6.

[0019] Furthermore, in step 3, IL-2, IL-7, or a combination thereof are added.

[0020] Furthermore, in step 4, the CAR molecule includes step cFv, hinge region, transmembrane region, co-stimulatory domain and CD3ζ signaling domain.

[0021] Furthermore, in step 5, during quality testing, cell viability must be ≥85% and CAR positivity rate must be ≥20%.

[0022] Furthermore, the following steps are also included:

[0023] Step 6: Digital marking of cleaning areas and pipelines:

[0024] The fully automated cell preparation instrument has a built-in fluid path database, which completes the labeling and parameter acquisition of the entire pipeline and area. The labeled objects include transfer pipelines shared by different fluids before and after, centrifuge cup interfaces, magnetic separator flow paths, mixing module chambers, liquid bag connectors, and dead volume dead angles; the parameters include the total length of the clean section pipeline. , inner diameter Total internal wall wetting area Total dead volume of each cleaned area; wettability coefficient of pipe material. Rated delivery flow rate of cleaning fluid ;

[0025] Labeling solutions include peripheral blood sample solutions, activation reagent solutions, viral vector solutions, cell culture media, and washing concentrates;

[0026] Step 7: Perform basic cleaning.

[0027] For pipelines or areas shared by two different fluids, marking is required. Before transferring the latter fluid, basic cleaning is necessary by injecting a reference cleaning solution into the marked area or pipeline using a peristaltic pump. ,in To ensure a safety margin, the cleaning fluid should be used at the rated flow rate. Complete 3 basic cycles to cover the entire inner wall of the pipeline and the dead volume.

[0028] Furthermore, the following steps are also included:

[0029] Step 8: Intermittently monitor the cleaning effect:

[0030] Sensor microspheres are added to the cleaning solution at preset cleaning time intervals. The microspheres flow with the laminar flow of the cleaning solution, traversing the entire cleaning area and detecting the signal of residual residue in the cleaning area in real time.

[0031] The photoelectric and electrochemical modules beside the pipeline read the microsphere signals and calculate the actual residual rate. And set the maximum security threshold. ;like This indicates that the cleaning standard has been met; if This indicates that the cleaning standards are not met;

[0032] Step 9, Dynamic Additional Cleaning:

[0033] like It will automatically calculate the amount of additional cleaning solution needed. Additional cleaning time Based on the calculation results, additional cleaning fluid is injected and a replenishment cycle is performed until the residue meets the standard.

[0034] Step 10: Recovery and reuse of sensor microspheres:

[0035] The cleaning fluid carrying the sensor microspheres is eventually discharged from the cleaning area and the end of the pipeline, at which point the sensor microspheres are recovered using a recovery module.

[0036] Furthermore, in step 7, the formula for the total baseline cleaning time during basic cleaning is as follows:

[0037]

[0038] in, This is the total time for the basic loop, which defaults to 3 times. This refers to the total time for intermittent delivery and detection of the sensing microspheres.

[0039] The formula for the total amount of standard cleaning solution used is as follows:

[0040]

[0041]

[0042] in, Basic volume; The minimum amount of base fluid required to completely wet the inner wall of the pipeline; The necessary amount of liquid to fill dead volume and eliminate cleaning dead corners; The replacement fluid volume for a single cycle ensures full coverage of the entire area; This is the final baseline cleaning fluid dosage, including a safety margin. This is the safety margin factor.

[0043] Furthermore, in step 9, , The calculation formula is as follows:

[0044]

[0045]

[0046]

[0047]

[0048] in, , To enable real-time detection of residual signal intensity by sensing microspheres; The initial residual signal intensity before cleaning is obtained by a detection sensor at a fixed location;

[0049] The maximum allowable residue rate; , This refers to the final actual amount of cleaning solution used and the cleaning time.

[0050] This invention provides a method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument, which has the following beneficial effects:

[0051] 1. This method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument relies on the instrument to complete the entire closed-loop automated preparation process, which can significantly shorten the preparation cycle of CAR-T cells, improve the timeliness of clinical treatment, and effectively reduce the risk of cell contamination by eliminating the need for manual operation throughout the process. At the same time, it eliminates product fluctuations caused by differences in human factors and the environment, ensures the standardization and consistency of cell preparation, and produces cells with excellent activity, which can significantly enhance the clinical application value and popularization of CAR-T therapy.

[0052] 2. This method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument achieves precise positioning of the cleaning target through digital marking of pipelines and cleaning areas. Combined with parameter calculation and real-time residue detection, a cleaning control logic is constructed, which can accurately regulate the amount of cleaning solution and cleaning time, avoiding insufficient cleaning or waste of resources, effectively blocking cross-contamination during the transfer of different solutions, and using recyclable and reusable sensor microspheres to complete residue monitoring and recycling, improving the automation level and economy of the cleaning process, and providing a stable guarantee for the aseptic safety and compliant operation of cell preparation. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the preparation process of the present invention;

[0054] Figure 2 This is a schematic diagram of the device's external structure according to the present invention;

[0055] Figure 3 This is a schematic diagram of the fluid circuit of the device of the present invention;

[0056] Figure 4 This is an image of a CAR-T cell on day 1 of the present invention;

[0057] Figure 5 This is an imaging image of CAR-T cells on day 2 of this invention;

[0058] Figure 6 This is an imaging image of a CAR-T cell on day 3 according to the present invention; Detailed Implementation

[0059] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0060] like Figures 1-3 As shown, where, Figure 3 In the diagram, 1-22 represent control valves. This invention provides a technical solution: a method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument. The fully automated cell preparation instrument is a closed, automated cell processing platform, comprising a separation module, a reaction module, a culture module, and a purification module, and includes the following steps:

[0061] Step 1: Isolation of peripheral blood mononuclear cells:

[0062] Scan the code to enter the patient's sample information, and connect 50mL of peripheral blood to the centrifugation module;

[0063] Asymmetric error-proof centrifuge cups were used to perform density gradient centrifugation at 3000 r / min, and PBMCs were obtained within 1 hour.

[0064] Solution transfer triggers cleaning: After the PBMC is transferred to the reaction module, it enters the cleaning process through the shared transfer pipeline, see steps 6-10 for details;

[0065] The separation of the PBMC (Personalized Power Module) includes the following steps:

[0066] Step 1: The Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup;

[0067] Step 2: Whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup;

[0068] Step 3: Centrifuge the Ficoll solution and whole blood in a centrifuge cup until the PBMCs are separated;

[0069] Step 4: Extract the intermediate layer from the separation cup through valve 16 and valve 4 into the intermediate liquid bag for later use;

[0070] Step 5: The cleaning solution flows from valve 1 through peristaltic pump 1 and then through valve 17 into the centrifuge cup and is mixed to clean the centrifuge cup;

[0071] Step 6: The cleaning waste liquid in the centrifuge cup is discharged from valve 16 through pump 1 and then through valve 10.

[0072] Step 7: The spare intermediate layer liquid flows from valve 4 through pump 1 and then through valve 16 into the centrifuge cup;

[0073] Step 8: The washing solution flows from valve 1 through peristaltic pump 1 and then through valve 17 into the centrifuge cup and is mixed to wash the cells inside the centrifuge cup.

[0074] Step 9: In centrifugal mode, the supernatant flows from valve 16 through pump 1 and then through valve 10 to remove the supernatant after cleaning.

[0075] Step 10: Under centrifugal conditions, the liquid flows from valve 17 through pump 1 and then through valve 10 to further remove the supernatant and achieve concentration.

[0076] Step 11: The resuspension solution flows from valve 6 through pump 1, through valve 17, and into the centrifuge cup to resuspend the cells;

[0077] Step 2, T cell activation and CAR vector transduction:

[0078] The fluid module will reset the cleaned pipeline and automatically transfer the PBMC to the reaction module;

[0079] Add anti-CD3 / CD28 magnetic beads (1:1), lentiviral vector (MOI=4), 37℃, Cultured for 14 hours under the specified conditions;

[0080] Solution transfer triggers cleaning: After the transduction is completed, the common tubing from the reaction module to the culture module enters the cleaning process;

[0081] Specifically, the first step: the virus reagent flows from valve 5 through pump 1, then through valve 16 into the centrifuge cup;

[0082] Step 2: Pre-set incubation time at a constant temperature;

[0083] Step 3: During the cultivation process, gas is supplied to the centrifuge cup at the set concentration through valve 18. The gas is carbon dioxide.

[0084] Step 3, CAR-T cell expansion:

[0085] The intelligent temperature control module operates at 37℃±0.1℃. Cultured with IL-2 and IL-7 factors for 48 hours;

[0086] A microscope camera monitors cell density in real time, and a color camera monitors the state of the culture medium.

[0087] Solution transfer triggers cleaning: After amplification is complete, the common tubing from the culture module to the purification module enters the cleaning process;

[0088] During CAR-T cell expansion, the tubing to be reused is cleaned and the liquid bag is adjusted. The cleaning solution flows from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup and is mixed to clean the centrifuge cup. The cleaning solution then flows from valve 16 through pump 1 and valve 10 to drain the waste cleaning solution from the centrifuge cup. The cleaning solution then flows from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup. The cleaning solution in the centrifuge cup flows from valve 16 through pump 1 and valve 2 to clean tubing 2, and is ready for use. The cleaning solution then flows from valve 16 through pump 1 and valve 10 to drain the waste cleaning solution from the centrifuge cup. The second bag is then replaced with culture medium.

[0089] Then perform the following operations:

[0090] Step 1: The culture medium flows from valve 2 through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the replenishment.

[0091] Step 2: When the culture volume in the centrifuge cup is large, the medium needs to be changed: In the centrifugation state, the medium flows from valve 16 through pump 1, through valve 10 to remove the supernatant of the culture medium, and then the culture medium flows from valve 2 through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the medium change.

[0092] Step 3: Under constant temperature of 37 degrees Celsius, carry out amplification culture by circulating the medium according to the set time nodes;

[0093] Step 4: During the cultivation process, gas is supplied to the centrifuge cup at the set concentration through valve 18. The gas is carbon dioxide.

[0094] Step 4, Washing and Concentrating:

[0095] The magnetic beads, free viruses, and metabolites were removed by the purification module, and CAR-T cells were obtained by concentration.

[0096] CAR molecules include step cFv, hinge region, transmembrane region, co-stimulatory domain and CD3ζ signaling domain;

[0097] Specifically, the first step is: the washing solution flows from valve 1 through pump 1, through valve 17, into the centrifuge cup and is mixed to wash the cells in the centrifuge cup;

[0098] Step 2: Under centrifugal conditions, the supernatant after cleaning flows from valve 16 through pump 1 and then through valve 10 to remove the supernatant after cleaning.

[0099] Step 3: Under centrifugal conditions, the supernatant is further removed by pump 1 through valve 17 and then through valve 10 to achieve concentration.

[0100] Step 4: The resuspension solution flows from valve 6 through pump 1, through valve 17, into the centrifuge cup, mixes well, and resuspends the cells.

[0101] Step 5: The cells in the centrifuge cup are collected into the liquid bag by passing through valve 16, pump 1, and valve 7.

[0102] Step 5, Quality Inspection:

[0103] Detect cell viability, CAR positivity rate, and sterility indicators;

[0104] In quality testing, cell viability must be ≥85% and CAR positivity rate must be ≥20%.

[0105] Steps 1 through 5 are all completed in a fully automated cell preparation system, with a total preparation cycle of 2-3 days; Figures 4-6 As shown;

[0106] Step 6: Digital marking of cleaning areas and pipelines:

[0107] The fully automated cell preparation instrument has a built-in fluid path database, which completes the labeling and parameter acquisition of the entire pipeline and area. The labeled objects include transfer pipelines shared by different fluids before and after, centrifuge cup interfaces, magnetic separator flow paths, mixing module chambers, liquid bag connectors, and dead volume dead angles; the parameters include the total length of the clean section pipeline. , inner diameter Total internal wall wetting area Total dead volume of each cleaned area; wettability coefficient of pipe material. Rated delivery flow rate of cleaning fluid ;

[0108] Labeling solutions include peripheral blood sample solutions, activation reagent solutions, viral vector solutions, cell culture media, and washing concentrates;

[0109] Step 7: Perform basic cleaning.

[0110] For pipelines or areas shared by two different fluids, marking is required. Before transferring the latter fluid, basic cleaning is necessary by injecting a reference cleaning solution into the marked area or pipeline using a peristaltic pump. ,in To ensure a safety margin, the cleaning fluid should be used at the rated flow rate. Complete 3 basic cycles to cover the entire inner wall of the pipeline and dead volume;

[0111] The formula for the total baseline cleaning time during basic cleaning is as follows:

[0112]

[0113] in, This is the total time for the basic loop, which defaults to 3 times. This refers to the total time for intermittent delivery and detection of the sensing microspheres.

[0114] The formula for the total amount of standard cleaning solution used is as follows:

[0115]

[0116]

[0117] in, Basic volume; The minimum amount of base fluid required to completely wet the inner wall of the pipeline; The necessary amount of liquid to fill dead volume and eliminate cleaning dead corners; The replacement fluid volume for a single cycle ensures full coverage of the entire area; This is the final baseline cleaning fluid dosage, including a safety margin. This is the safety margin factor;

[0118] Step 8: Intermittently monitor the cleaning effect:

[0119] Sensor microspheres are added to the cleaning solution at preset cleaning time intervals. The microspheres flow with the laminar flow of the cleaning solution, traversing the entire cleaning area and detecting the signal of residual residue in the cleaning area in real time.

[0120] The photoelectric and electrochemical modules beside the pipeline read the microsphere signals and calculate the actual residual rate. And set the maximum security threshold. ;like This indicates that the cleaning standard has been met; if This indicates that the cleaning standards are not met;

[0121] Step 9, Dynamic Additional Cleaning:

[0122] like It will automatically calculate the amount of additional cleaning solution needed. Additional cleaning time Based on the calculation results, additional cleaning fluid is injected and a replenishment cycle is performed until the residue meets the standard.

[0123] , The calculation formula is as follows:

[0124]

[0125]

[0126]

[0127]

[0128] in, , To enable real-time detection of residual signal intensity by sensing microspheres; The initial residual signal intensity before cleaning is obtained by a detection sensor at a fixed location;

[0129] The maximum allowable residue rate; , This refers to the final actual amount of cleaning solution used and the cleaning time.

[0130] Step 10: Recovery and reuse of sensor microspheres:

[0131] The cleaning fluid carrying the sensing microspheres is finally discharged from the cleaning area and the end of the pipeline. At this time, the sensing microspheres are recovered using a recovery module. The sensing microspheres are VDO Biotech flow cytometry fluorescent magnetic microspheres (model: MFB100518A).

[0132] The specific instructions for steps 6-10 are as follows:

[0133] First, the control module calculates the baseline cleaning fluid usage and baseline cleaning time in real time based on the pre-modeling parameters; then, the fluid module drives the peristaltic pump to inject the baseline cleaning fluid into the marked cleaning area and pipelines. The cleaning fluid is applied at the rated flow rate. Complete 3 basic cycles to cover the entire inner wall of the pipeline and dead volume;

[0134] VDO Biotech flow cytometry fluorescent magnetic microspheres (model: MFB100518A) were selected. Specific parameters are as follows:

[0135] Particle size: 4μm (uniformity CV≤5%), density 1.05g / cm³ (matched with cleaning solution to ensure laminar flow following);

[0136] Core material: Core-shell structure (superparamagnetic, saturation magnetization 18 emu / g);

[0137] Surface modification: Carboxyl (-COOH) group, which can be coupled to protein / virus-specific fluorescent probes (APC fluorescent channel).

[0138] Compliance: GMP-grade production, endotoxin <0.05 EU / mL, no DNA / RNase contamination;

[0139] The sensor microspheres were added in three separate administrations at time intervals of 0, 5 min, and 10 min (each administration containing 1.2 × 10⁻⁶ microspheres). 6 (One microsphere); the microspheres flow with the laminar flow of the cleaning fluid, traversing the entire cleaning area, and specifically bind to residual substances through surface fluorescent probes, outputting detection signals in real time;

[0140] The pipeline-side photoelectric detection module (detection wavelength 660 nm) reads the fluorescence signal intensity of the microspheres and calculates the actual residual rate according to the formula. ;like This indicates that the cleaning standard has been met; if This indicates that the cleaning standards are not met; if It will automatically calculate the amount of additional cleaning solution needed. Additional cleaning time Based on the calculation results, additional cleaning solution is injected, and a replenishment cycle is performed until the residue meets the standard (microsphere signal display). );

[0141] The fluid module introduces the cleaning solution containing microspheres into the recovery module for recycling.

[0142] In summary, this method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument achieves a completely closed and automated preparation process, which can significantly shorten the preparation cycle of CAR-T cells, improve the timeliness of clinical treatment, and effectively reduce the risk of cell contamination by eliminating the need for manual operation throughout the process. At the same time, it eliminates product fluctuations caused by differences in human factors and the environment, ensures the standardization and consistency of cell preparation, and produces cells with excellent activity, which can significantly enhance the clinical application value and popularity of CAR-T therapy.

[0143] Furthermore, by digitally marking pipelines and clean areas, the cleaning target can be accurately located. Combined with parameter calculation and real-time residue detection, a closed-loop control logic for cleaning can be constructed, which can accurately regulate the amount of cleaning solution and the cleaning time, avoid insufficient cleaning or waste of resources, effectively block cross-contamination during the transfer of different solutions, and complete residue monitoring and recycling with recyclable and reusable sensor microspheres, thereby improving the automation level and economy of the cleaning process and providing a stable guarantee for the aseptic safety and compliant operation of cell preparation.

[0144] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument, characterized in that: Includes the following steps: Step 1: Isolation of peripheral blood mononuclear cells: The patient's peripheral blood sample was added to the separation module of the fully automated cell preparation instrument, and PBMCs were obtained by density gradient centrifugation. PBMC separation involves the following steps: Step 1: The Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; Step 2: Whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; Step 3: Centrifuge the Ficoll solution and whole blood in a centrifuge cup until the PBMCs are separated; Step 4: Extract the intermediate layer from the separation cup through valve 16 and valve 4 into the intermediate liquid bag for later use; Step 5: The cleaning solution flows from valve 1 through peristaltic pump 1 and then through valve 17 into the centrifuge cup and is mixed to clean the centrifuge cup; Step 6: The cleaning waste liquid in the centrifuge cup is discharged from valve 16 through pump 1 and then through valve 10. Step 7: The spare intermediate layer liquid flows from valve 4 through pump 1 and then through valve 16 into the centrifuge cup; Step 8: The washing solution flows from valve 1 through peristaltic pump 1 and then through valve 17 into the centrifuge cup and is mixed to wash the cells inside the centrifuge cup. Step 9: In centrifugal mode, the supernatant flows from valve 16 through pump 1 and then through valve 10 to remove the supernatant after cleaning. Step 10: Under centrifugal conditions, the liquid flows from valve 17 through pump 1 and then through valve 10 to further remove the supernatant and achieve concentration. Step 11: The resuspension solution flows from valve 6 through pump 1, through valve 17, and into the centrifuge cup to resuspend the cells; Step 2, T cell activation and CAR vector transduction: PBMCs were transferred into a reaction module, and activating reagents and viral vectors carrying CAR sequences were added for simultaneous activation and transduction. Specifically, the first step: the virus reagent flows from valve 5 through pump 1, then through valve 16 into the centrifuge cup; Step 2: Pre-set incubation time at a constant temperature; Step 3: During the cultivation process, gas is supplied to the centrifuge cup at the set concentration through valve 18. The gas is carbon dioxide. Step 3, CAR-T cell expansion: In the culture module, cells are cultured for 1-2 days under preset temperature, gas concentration, and cytokine conditions; Step 4, Washing and Concentrating: The magnetic beads, free viruses, and metabolites were removed by the purification module, and CAR-T cells were obtained by concentration. Step 5, Quality Inspection: Detect cell viability, CAR positivity rate, and sterility indicators; Steps 1 through 5 are all completed in a fully automated cell preparation instrument, with a total preparation cycle of 2-3 days.

2. The method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step 2, the activating agent is anti-CD3 / CD28 magnetic beads, and the ratio of magnetic beads to cells is 1:1 to 2:

1.

3. The method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step 2, the viral vector is a lentivirus or a retrovirus with an MOI value of 2-6.

4. The method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step 3, IL-2, IL-7, or a combination thereof are added. During CAR-T cell expansion, the tubing to be reused is cleaned and the liquid bag is adjusted. The cleaning solution flows from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup and is mixed to clean the centrifuge cup. The cleaning solution then flows from valve 16 through pump 1 and valve 10 to drain the waste cleaning solution from the centrifuge cup. The cleaning solution then flows from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup. The cleaning solution in the centrifuge cup flows from valve 16 through pump 1 and valve 2 to clean tubing 2, and is ready for use. The cleaning solution then flows from valve 16 through pump 1 and valve 10 to drain the waste cleaning solution from the centrifuge cup. The second bag is then replaced with culture medium. Then perform the following operations: Step 1: The culture medium flows from valve 2 through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the replenishment. Step 2: When the culture volume in the centrifuge cup is large, the medium needs to be changed: In the centrifugation state, the medium flows from valve 16 through pump 1, through valve 10 to remove the supernatant of the culture medium, and then the culture medium flows from valve 2 through pump 1, through valve 15, through the preheating module, and into the centrifuge cup to complete the medium change. Step 3: Under constant temperature of 37 degrees Celsius, carry out amplification culture by circulating the medium according to the set time nodes; Step 4: During the cultivation process, gas is supplied to the centrifuge cup at the set concentration through valve 18. The gas is carbon dioxide.

5. A method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step 4, the CAR molecule includes step cFv, hinge region, transmembrane region, co-stimulatory domain and CD3ζ signaling domain; During washing and concentration, specifically, the first step is: the washing solution flows from valve 1 through pump 1, through valve 17, into the centrifuge cup and is mixed to wash the cells in the centrifuge cup. Step 2: Under centrifugal conditions, the supernatant after cleaning flows from valve 16 through pump 1 and then through valve 10 to remove the supernatant after cleaning. Step 3: Under centrifugal conditions, the supernatant is further removed by pump 1 through valve 17 and then through valve 10 to achieve concentration. Step 4: The resuspension solution flows from valve 6 through pump 1, through valve 17, into the centrifuge cup, mixes well, and resuspends the cells. Step 5: The cells in the centrifuge cup are collected into the liquid bag by passing through valve 16, pump 1, and valve 7.

6. The method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step 5, during quality testing, cell viability must be ≥85% and CAR positivity rate must be ≥20%.

7. A method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: It also includes the following steps: Step 6: Digital marking of cleaning areas and pipelines: The fully automated cell preparation instrument has a built-in fluid path database, which completes the labeling and parameter acquisition of the entire pipeline and area. The labeled objects include transfer pipelines shared by different fluids before and after, centrifuge cup interfaces, magnetic separator flow paths, mixing module chambers, liquid bag connectors, and dead volume dead angles; the parameters include the total length of the clean section pipeline. , inner diameter Total internal wall wetting area Total dead volume of each cleaned area; wettability coefficient of pipe material. Rated delivery flow rate of cleaning fluid ; Labeling solutions include peripheral blood sample solutions, activation reagent solutions, viral vector solutions, cell culture media, and washing concentrates; Step 7: Perform basic cleaning. For pipelines or areas shared by two different fluids, marking is required. Before transferring the latter fluid, basic cleaning is necessary by injecting a reference cleaning solution into the marked area or pipeline using a peristaltic pump. ,in To ensure a safety margin, the cleaning fluid should be used at the rated flow rate. Complete 3 basic cycles to cover the entire inner wall of the pipeline and the dead volume.

8. A method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 7, characterized in that: It also includes the following steps: Step 8: Intermittently monitor the cleaning effect: Sensor microspheres are added to the cleaning solution at preset cleaning time intervals. The microspheres flow with the laminar flow of the cleaning solution, traversing the entire cleaning area and detecting the signal of residual residue in the cleaning area in real time. The photoelectric and electrochemical modules beside the pipeline read the microsphere signals and calculate the actual residual rate. And set the maximum security threshold. ;like This indicates that the cleaning standard has been met; if This indicates that the cleaning standards are not met; Step 9, Dynamic Additional Cleaning: like It will automatically calculate the amount of additional cleaning solution needed. Additional cleaning time Based on the calculation results, additional cleaning fluid is injected and a replenishment cycle is performed until the residue meets the standard. Step 10: Recovery and reuse of sensor microspheres: The cleaning fluid carrying the sensor microspheres is eventually discharged from the cleaning area and the end of the pipeline, at which point the sensor microspheres are recovered using a recovery module.

9. A method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 6, characterized in that: In step 7, the formula for the total baseline cleaning time during basic cleaning is as follows: ; in, This is the total time for the basic loop, which defaults to 3 times. This refers to the total time for intermittent delivery and detection of the sensing microspheres. The formula for the total amount of standard cleaning solution used is as follows: ; ; in, Basic volume; The minimum amount of base fluid required to completely wet the inner wall of the pipeline; The necessary amount of liquid to fill dead volume and eliminate cleaning dead corners; The replacement fluid volume for a single cycle ensures full coverage of the entire area; This is the final baseline cleaning fluid dosage, including a safety margin. This is the safety margin factor.

10. A method for rapidly preparing CAR-T cells using a fully automated cell preparation instrument according to claim 8, characterized in that: In step 9 , The calculation formula is as follows: ; ; ; ; in, , To enable real-time detection of residual signal intensity by sensing microspheres; The initial residual signal intensity before cleaning is obtained by a detection sensor at a fixed location; The maximum allowable residue rate; , This refers to the final actual amount of cleaning solution used and the cleaning time.

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