A method for preparing CAR-T cells using a fully automated cell preparator

Abstract

This invention discloses a method for preparing CAR-T cells using a fully automated cell preparation instrument, relating to the fields of bioengineering and cell therapy technology. The method includes the following steps: S1, sample introduction and pretreatment; S2, PBMC separation; S3, cell incubation and activation; S4, cell sorting; S5, lentivirus transduction; S6, amplification culture; S7, cell harvesting; and S8, formulation dispensing. This invention, through fully automated closed-loop operation and parameter-responsive adaptive centrifugation control, significantly reduces human error and the risk of exogenous contamination, achieving precise dynamic adaptation of key PBMC separation process parameters. This significantly improves cell separation purity, recovery rate, and viability, ensuring uniform and stable cell product quality, and fully meeting the compliance and practical requirements for the industrial-scale preparation of CAR-T cells.

Description

Technical Field

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

[0002] CAR-T cell therapy, a revolutionary technology in tumor immunotherapy, has shown significant efficacy in the treatment of diseases such as hematological malignancies. Its core is to modify the patient's T cells through genetic engineering, enabling them to specifically recognize and kill tumor cells.

[0003] Although some automated cell processing equipment has emerged in the existing technology, it has limited ability to dynamically control key process parameters during centrifugation, making it difficult to meet the GMP compliance requirements and large-scale production needs of CAR-T cell preparation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for 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 preparing CAR-T cells using a fully automated cell preparation instrument, comprising the following steps:

[0006] S1. Sample Import and Preprocessing:

[0007] Peripheral blood samples or Leukopak samples are injected into the sample chamber of the fully automated cell preparation instrument, and the equipment automatically detects the sample volume and cell concentration;

[0008] S2 and PBMC separation:

[0009] PBMCs are automatically separated and washed using density gradient centrifugation.

[0010] Specifically: Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; Ficoll solution and whole blood are centrifuged in the centrifuge cup until PBMCs are separated.

[0011] Extract the intermediate layer from the separation cup through valve 16 and then through valve 4 into the intermediate liquid bag for later use.

[0012] 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; the cleaning waste solution in the centrifuge cup is discharged from valve 16 through pump 1 and then through valve 10.

[0013] The spare intermediate liquid flows from valve 4 through pump 1 and then through valve 16 into the centrifuge cup;

[0014] 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 cells inside the centrifuge cup.

[0015] During centrifugation, the supernatant flows from valve 16 through pump 1 and then through valve 10 to remove the supernatant after washing; during centrifugation, the supernatant flows from valve 17 through pump 1 and then through valve 10 to further remove the supernatant and achieve concentration; the resuspension flows from valve 6 through pump 1 and then through valve 17 into the centrifuge cup to resuspend the cells.

[0016] S3, Cell incubation and activation:

[0017] PBMCs were placed in a cell incubation module and activated in a culture medium containing CD3 / CD28 antibody and IL-2;

[0018] S4. Cell sorting:

[0019] Enrichment by magnetic bead sorting cell;

[0020] Specifically: First, magnetic beads are labeled and flow from valve 5 through pump 1, then through valve 16 into the centrifuge cup; the cells are incubated at a constant temperature of 37 degrees Celsius for a set time; washing solution flows from valve 1 through pump 1, then through valve 17 into the centrifuge cup and is mixed to wash the cells; during centrifugation, the supernatant is removed from valve 16 through pump 1, then through valve 10; during centrifugation, the supernatant is further removed from valve 17 through pump 1, then through valve 10, achieving concentration; resuspension solution flows from valve 6 through pump 1, then through valve 17 into the centrifuge cup to resuspend the cells; finally, the labeled cells are collected from valve 16 through pump 1, then through valve 7. Collect the cells into the liquid bag; turn on the magnet at magnetic column C2; labeled cells flow from valve 8 through magnetic column C1, then through magnetic column C2, then through pump 2, and through valve 22 for cell sorting. During this process, positively labeled cells are adsorbed onto magnetic column C2, while negative cells flow into the negative cell bag; buffer solution flows from valve 9 through magnetic column C1, then through magnetic column C2, then through pump 2, and through valve 22, rinsing all negative cells into the negative cell bag; turn off the magnet at magnetic column C2; culture medium flows from valve 2 through valve 11, then through magnetic column C2, then through pump 2, rinsing the positively labeled cells on magnetic column C2, and flowing through valve 20 into the positive cell collection bag;

[0021] S5, Lentiviral transduction:

[0022] The sorted T cells were incubated with a lentiviral vector carrying the CAR gene in a transduction module.

[0023] The first step involves cleaning the pipelines that need to be reused and adjusting the liquid bags. The specific steps are as follows:

[0024] 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.

[0025] The cleaning waste liquid in the centrifuge cup is discharged from valve 16 through pump 1 and then through valve 10.

[0026] The cleaning solution flows from valve 1 through peristaltic pump 1 and then through valve 17 into the centrifuge cup;

[0027] The cleaning solution in the centrifuge cup flows from valve 16 through pump 1 and then through valve 5 to clean pipeline 5, and is ready for use.

[0028] The cleaning waste liquid in the centrifuge cup is discharged from valve 16 through pump 1 and then through valve 10.

[0029] Replace bag #5 with lentivirus or retrovirus;

[0030] The specific process for lentivirus transduction is as follows:

[0031] The virus reagent flows from valve 5 through pump 1, then through valve 16 into the centrifuge cup to begin transduction; and the centrifuge cup is kept at a constant temperature of 37 degrees Celsius for activation culture; during the culture process, gas is supplied to the centrifuge cup at the set concentration through valve 18.

[0032] S6. Amplification and Culture:

[0033] Perfusion culture is performed in the amplification culture module, automatically maintaining nutrient and environmental parameters;

[0034] Specifically: The culture medium flows from valve 2 through pump 1 to valve 15, then through the preheating module and into the centrifuge cup, thus completing the replenishment; when the culture volume in the centrifuge cup is greater than the preset value, the medium needs to be changed: in centrifugation mode, the culture medium flows from valve 16 through pump 1 to valve 10 to remove the supernatant, and then the culture medium flows from valve 2 through pump 1 to valve 15, through the preheating module and into the centrifuge cup, thus completing the medium change;

[0035] Under constant temperature of 37 degrees Celsius, the amplification culture was carried out according to the preset time node liquid exchange steps; during the culture process, gas was supplied to the centrifuge cup at a preset concentration through valve No. 18, and the gas was carbon dioxide.

[0036] S7. Cell Harvesting:

[0037] CAR-T cells were collected by centrifugation and concentration.

[0038] S8. Formulation repackaging:

[0039] Cells are resuspended in formulation buffer and automatically dispensed into cryopreservation bags or infusion bags;

[0040] Specifically: the cryopreservation solution flows from valve 9 through pump 2, then through valve 19, then through valve 14, and finally through valve 2 into the mixing bag; the mixing plate in the cooling chamber thoroughly mixes the mixing bag; the mixed cell solution flows from valve 2 through pump 1, through valve 12, and is dispensed into cryopreservation bags according to the set dosage, completing the dispensing bag by bag.

[0041] All of the above steps are completed in a fully automated cell preparation instrument.

[0042] Furthermore, in step S2, a microscope camera is used to acquire cell images in real time through the observation hole at the bottom of the centrifuge cup, automatically identifying and counting cells and accurately calculating cell density; a color camera is used to monitor changes in the culture medium color and gradient interface status in real time; and the following parameter-responsive centrifugation process is implemented in conjunction with each component:

[0043] 1) Adaptive calculation of centrifugal force:

[0044]

[0045] In the formula, It is centrifugal force; The real-time sedimentation rate of cells is identified by a microscope camera; The baseline settlement rate; Centrifugal force as reference; centrifugal force adjustment coefficient The expression used to regulate the relationship between the intensity and time of cell treatment during centrifugation is: The calculation results are constrained to a safe range using clamping functions.

[0046]

[0047] 2) Adaptive calculation of centrifugation time:

[0048]

[0049] In the formula, Centrifugation time; The reference centrifugation time; Target PBMC layer thickness; The real-time thickness of the PBMC layer is identified by a color camera. Gradient interface sharpness is determined by a color camera; time adjustment factor. The calculation results are constrained to a safe range using a clamping function:

[0050]

[0051] 3) Temperature control adaptive calculation:

[0052]

[0053] In the formula, For temperature; Reference temperature; Real-time PBMC viability, obtained from microscopic camera image analysis; temperature regulation coefficient. The calculation results are constrained to a safe range using a clamping function:

[0054]

[0055] 4) Adaptive calculation of acceleration / deceleration slope:

[0056]

[0057]

[0058] In the formula, For interface perturbation; Accelerate the slope as a baseline; The reference deceleration slope; To accelerate the slope; The deceleration slope; the slope attenuation coefficient. The calculation result enforces lower limit protection, that is: , .

[0059] Furthermore, based on the calculated parameters of formulas 1), 2), 3), and 4) above, the servo motor performs density gradient centrifugation according to the sequence of "stepwise acceleration → constant temperature and constant force centrifugation → stepwise deceleration." During centrifugation, the density gradient medium is Ficoll-PaquePlus with a density of 1.077 g / mL, automatically forming a four-layer structure: plasma layer → PBMC layer → medium layer → red blood cell layer. After centrifugation, a sterile aspiration needle is inserted along the inner wall of the centrifuge cup, and segmented flow rate aspiration is performed according to the cell density of the aspiration segment.

[0060]

[0061] In the formula, The real-time cell density of the aspirated segment was counted using a microscope camera. The suction flow rate is denoted as .

[0062] Furthermore, in step S3, the concentration of CD3 antibody is 1.8–2.2 μg / ml, the concentration of CD28 antibody is 1.8–2.2 μg / ml, the concentration of IL-2 is 200 U / ml, and the incubation time is 48 h.

[0063] Furthermore, in step S4, the purity of the sorted T cells is ≥90%.

[0064] Furthermore, in step S5, the MOI value of lentivirus transduction is 1–5, and the transduction time is 12h.

[0065] Furthermore, in step S6, the amplification culture uses serum-free medium, the glucose concentration is maintained at 5–10 mmol / L, the lactate concentration is <15 mmol / L, and the culture time is 5–7 days.

[0066] Furthermore, in step S8, the concentration of the cell preparation is... The repackaged volume is 10–50 ml / bag.

[0067] Furthermore, the CAR gene target is selected from at least one of CD19, CD20, BCMA, CD22, CD30, CD33, HER2, EGFRvIII, or PSMA.

[0068] This invention provides a method for preparing CAR-T cells using a fully automated cell preparation instrument, which has the following beneficial effects:

[0069] 1. This method for preparing CAR-T cells using a fully automated cell preparation instrument effectively solves the pain points of traditional CAR-T cell preparation, such as high dependence on manual labor, high risk of contamination, poor product quality stability, low preparation efficiency, and insufficient dynamic control capability of key process parameters of existing automated equipment, making it unsuitable for GMP compliance and large-scale production. Through fully closed automated operation and parameter-responsive adaptive centrifugation control, it significantly reduces human operation errors and the risk of exogenous contamination, achieves precise dynamic adaptation of key process parameters for PBMC separation, significantly improves cell separation purity, recovery rate and viability, ensures uniform and stable cell product quality, and fully meets the compliance and practical requirements of industrial and large-scale CAR-T cell preparation. Attached Figure Description

[0070] Figure 1 This is a detailed flowchart illustrating the steps of the present invention;

[0071] Figure 2 This is a schematic diagram of the device's external structure according to the present invention;

[0072] Figure 3 This is a schematic diagram of the liquid circuit of the present invention;

[0073] Figure 4 This is an image of a CART cell on day 3 according to the present invention;

[0074] Figure 5 This is an image of a CART cell on day 5 according to the present invention;

[0075] Figure 6This is an image of a CART cell on day 7 according to the present invention; Detailed Implementation

[0076] 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.

[0077] 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 preparing CAR-T cells using a fully automated cell preparation instrument, comprising the following steps:

[0078] S1. Sample Import and Preprocessing:

[0079] Peripheral blood samples or Leukopak samples are injected into the sample chamber of the fully automated cell preparation instrument. The equipment automatically performs anticoagulation treatment and detects basic parameters such as sample volume and cell concentration.

[0080] S2 and PBMC separation:

[0081] The PBMC separation module is activated. This module includes a drive unit, centrifuge cups, a rotating base, a microscope camera, and a color camera. The centrifuge cups and rotating base employ an asymmetrical, foolproof design, achieving unidirectional assembly through the asymmetrical shape of the structure itself. After static balance verification, the center of gravity offset is controlled within 0.1 mm. The drive unit uses a servo motor to drive the rotating base, enabling the centrifuge cup to rotate at speeds ranging from 10 to 5000 rpm. At low speeds (10-100 rpm), it also functions as a cell suspension mixer. The microscope camera acquires cell images in real time through an observation port at the bottom of the centrifuge cup, automatically identifying and counting cells and accurately calculating cell density. The color camera monitors changes in culture medium color and gradient interface status in real time. All components work together to execute the following parameter-responsive centrifugation process:

[0082] 1) Adaptive calculation of centrifugal force:

[0083]

[0084] In the formula, It is centrifugal force; The real-time sedimentation rate of cells is identified by a microscope camera; The baseline settlement rate; Centrifugal force as reference; centrifugal force adjustment coefficient The expression used to regulate the relationship between the intensity and time of cell treatment during centrifugation is: The calculation results are constrained to a safe range using clamping functions.

[0085]

[0086] Right now ;

[0087] 2) Adaptive calculation of centrifugation time:

[0088]

[0089] In the formula, Centrifugation time; The reference centrifugation time; Target PBMC layer thickness; The real-time thickness of the PBMC layer is identified by a color camera. Gradient interface sharpness is determined by a color camera; time adjustment factor. The calculation results are constrained to a safe range using a clamping function:

[0090]

[0091] Right now ;

[0092] 3) Temperature control adaptive calculation:

[0093]

[0094] In the formula, For temperature; Reference temperature; Real-time PBMC viability, obtained from microscopic camera image analysis; temperature regulation coefficient. The calculation results are constrained to a safe range using a clamping function:

[0095]

[0096] Right now ;

[0097] 4) Adaptive calculation of acceleration / deceleration slope:

[0098]

[0099]

[0100] In the formula, For interface perturbation; Accelerate the slope as a baseline; The reference deceleration slope; To accelerate the slope; The deceleration slope; the slope attenuation coefficient. The calculation results enforce lower limit protection.

[0101] Right now: , ;

[0102] Based on the calculated parameters of formulas 1), 2), 3), and 4) above, the servo motor performs density gradient centrifugation according to the sequence of "stepwise acceleration → constant temperature and constant force centrifugation → stepwise deceleration". During centrifugation, the density gradient medium is Ficoll-Paque Plus with a density of 1.077 g / mL, automatically forming a four-layer structure: plasma layer → PBMC layer → medium layer → red blood cell layer. After centrifugation, a sterile aspiration needle is inserted along the inner wall of the centrifuge cup, and segmented flow rate aspiration is performed according to the cell density of the aspiration segment.

[0103]

[0104] In the formula, The real-time cell density of the aspirated segment was counted using a microscope camera. The suction flow rate; and ;

[0105] Based on cell density Segmented regulation, The flow rate was 1.2 mL / s. The flow rate was 1.0 mL / s. 0.3~1.0 mL / s;

[0106] S3, Cell incubation and activation:

[0107] After washing, PBMC cells were transferred to the cell incubation module. The device automatically added activation medium containing CD3 monoclonal antibody (2±0.2μg / ml), CD28 monoclonal antibody (2±0.2μg / ml), and IL-2 (200U / ml), and incubated at 37℃. Incubate for 48 hours in the specified environment to complete T cell activation;

[0108] S4. Cell sorting:

[0109] The activated cell suspension enters the cell sorting module, where it is specifically enriched using magnetic bead sorting technology. Cells, after sorting, have a cell purity of ≥90%;

[0110] S5, Lentiviral transduction:

[0111] After sorting, T cells are transferred to a lentiviral transduction module. The device automatically adds a lentiviral vector carrying a CAR gene targeting a specific antigen, controlling the MOI value to 1-5, and maintaining the temperature at 37°C. The cells were incubated for 12 hours under the specified conditions, and the transfection status was observed in real time through the built-in cell digital monitoring unit during the transduction process.

[0112] S6. Amplification and Culture:

[0113] After transduction, the cells were transferred to an expansion culture module and perfused in serum-free medium. The equipment monitored the medium consumption in real time using an image acquisition device, automatically replenishing nutrients to maintain a glucose concentration of 5-10 mmol / L and a lactate concentration of <15 mmol / L in the culture system at 37°C. Cultured in an environmentally friendly environment for 5-7 days, allowing CAR-T cells to expand to 1×10⁻⁶ cells. 9 above;

[0114] S7. Cell Harvesting:

[0115] After amplification, the cell harvesting module automatically performs centrifugation (500g centrifugation for 10min) to remove supernatant and metabolic waste, and collect highly active CAR-T cell pellet.

[0116] S8. Formulation repackaging:

[0117] Resuspend the cell pellet in the formulation buffer and adjust the cell concentration to [the desired level]. The preparation is automatically dispensed into sterile cryopreservation bags or infusion bags through the formulation dispensing module, with each bag having a volume of 10-50ml, thus completing the preparation.

[0118] The CAR gene target is selected from at least one of CD19, CD20, BCMA, CD22, CD30, CD33, HER2, EGFRvIII or PSMA; all steps of the device are performed in a closed sterile environment, achieving full automation and traceability.

[0119] Example 1: Preparation of anti-CD19 CAR-T cells;

[0120] Sample preparation: 50ml of peripheral blood from a healthy donor, 50ml of equipment detection volume, cell concentration... .

[0121] PBMC separation: Automated density gradient centrifugation was used to collect approximately [number missing] PBMCs. There were 100 individuals, with a survival rate of 98%.

[0122] Cell activation: Add activation medium to 200 ml, add CD3 (2 μg / ml), CD28 (2 μg / ml), and IL-2 (200 U / ml), incubate at 37°C. Incubate for 48 hours.

[0123] Cell sorting: obtained by magnetic bead sorting cell One, purity 92.3%.

[0124] Lentiviral transduction: CD19-CAR lentivirus was added, MOI=3, incubated for 12h, and the transduction efficiency was 73.5%.

[0125] Amplification culture: Serum-free perfusion culture for 6 days, total cell count The survival rate was 96.2%.

[0126] Harvesting and Packaging: Resuspended to 10ml / bag, a total of 6 bags of finished product and half a bag of quality inspection sample.

[0127] Example 2: Preparation of anti-BCMA CAR-T cells;

[0128] Sample preparation: 200ml of Leukopak from multiple myeloma patients, cell concentration... .

[0129] PBMC separation: PBMCs were collected by density gradient centrifugation. One, survival rate 95%.

[0130] Cell activation: Add culture medium to a final volume of 500 ml, then add CD3 (1.8 μg / ml), CD28 (2.2 μg / ml), and IL-2 (200 U / ml), and incubate at 37°C. Incubate for 48 hours.

[0131] Cell sorting: Magnetic bead sorting Cells, 94.7% purity, harvested indivual.

[0132] Lentiviral transduction: BCMA-CAR lentivirus was added, MOI=4, and incubated for 12h, with a transduction efficiency of 65.2%.

[0133] Expansion culture: Perfusion culture for 7 days, total cell count Survival rate 95%, such as Figures 4-6 As shown, Figures 4-6 This is a schematic diagram of CART cells on days 3, 5, and 7.

[0134] Harvesting and Packaging: Adjusting Concentration 20ml / bag, a total of 6 bags of finished product and half a bag of quality inspection sample.

[0135] Based on the above description, this invention effectively solves the pain points of traditional CAR-T cell preparation, such as high dependence on manual labor, high risk of contamination, poor product quality stability, low preparation efficiency, insufficient dynamic control capability of key process parameters of existing automated equipment, and inability to adapt to GMP compliance and large-scale production. Through fully closed automated operation and parameter-responsive adaptive centrifugation control, it significantly reduces human operation errors and the risk of exogenous contamination, achieves precise dynamic adaptation of key process parameters for PBMC separation, significantly improves cell separation purity, recovery rate and viability, ensures uniform and stable cell product quality, and fully meets the compliance and practical requirements of industrial and large-scale CAR-T cell preparation.

[0136] 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 preparing CAR-T cells using a fully automated cell preparator, characterized by: Includes the following steps: S1. Sample Import and Preprocessing: Peripheral blood samples or Leukopak samples are injected into the sample chamber of the fully automated cell preparation instrument, and the equipment automatically detects the sample volume and cell concentration; S2 and PBMC separation: PBMCs are automatically separated and washed using density gradient centrifugation. Specifically: Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup; whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup. Centrifuge Ficoll solution and whole blood in a centrifuge cup until PBMCs are separated; Extract the intermediate layer from the separation cup through valve 16 and then through valve 4 into the intermediate liquid bag for later use. 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; the cleaning waste solution in the centrifuge cup is discharged from valve 16 through pump 1 and then through valve 10. The spare intermediate liquid flows from valve 4 through pump 1 and then through valve 16 into the centrifuge cup; 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 cells inside the centrifuge cup. During centrifugation, the supernatant flows from valve 16 through pump 1 and then through valve 10 to remove the supernatant after washing; during centrifugation, the supernatant flows from valve 17 through pump 1 and then through valve 10 to further remove the supernatant and achieve concentration; the resuspension flows from valve 6 through pump 1 and then through valve 17 into the centrifuge cup to resuspend the cells. S3, Cell incubation and activation: PBMCs were placed in a cell incubation module and activated in a culture medium containing CD3 / CD28 antibody and IL-2; S4. Cell sorting: Enrichment by magnetic bead sorting cells; Specifically: First, magnetic beads are labeled and flow from valve 5 through pump 1, then through valve 16 into the centrifuge cup; the cells are incubated at a constant temperature of 37 degrees Celsius for a set time; washing solution flows from valve 1 through pump 1, then through valve 17 into the centrifuge cup and is mixed to wash the cells; during centrifugation, the supernatant is removed from valve 16 through pump 1, then through valve 10; during centrifugation, the supernatant is further removed from valve 17 through pump 1, then through valve 10, achieving concentration; resuspension solution flows from valve 6 through pump 1, then through valve 17 into the centrifuge cup to resuspend the cells; finally, the labeled cells are collected from valve 16 through pump 1, then through valve 7. Collect the cells into the liquid bag; turn on the magnet at magnetic column C2; labeled cells flow from valve 8 through magnetic column C1, then through magnetic column C2, then through pump 2, and through valve 22 for cell sorting. During this process, positively labeled cells are adsorbed onto magnetic column C2, while negative cells flow into the negative cell bag; buffer solution flows from valve 9 through magnetic column C1, then through magnetic column C2, then through pump 2, and through valve 22, rinsing all negative cells into the negative cell bag; turn off the magnet at magnetic column C2; culture medium flows from valve 2 through valve 11, then through magnetic column C2, then through pump 2, rinsing the positively labeled cells on magnetic column C2, and flowing through valve 20 into the positive cell collection bag; S5, Lentiviral transduction: The sorted T cells were incubated with a lentiviral vector carrying the CAR gene in a transduction module. S6. Amplification and Culture: Perfusion culture is performed in the amplification culture module, automatically maintaining nutrient and environmental parameters; Under constant temperature of 37 degrees Celsius, the amplification culture was carried out according to the preset time node liquid exchange steps; during the culture process, gas was supplied to the centrifuge cup at a preset concentration through valve No. 18, and the gas was carbon dioxide. S7. Cell Harvesting: CAR-T cells were collected by centrifugation and concentration. S8. Formulation repackaging: Cells are resuspended in formulation buffer and automatically dispensed into cryopreservation bags or infusion bags; All of the above steps are completed in a fully automated cell preparation instrument.

2. The method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S2, a microscope camera is used to acquire cell images in real time through the observation hole at the bottom of the centrifuge cup, automatically identifying and counting cells and accurately calculating cell density; a color camera is used to monitor changes in the culture medium color and gradient interface status in real time; and all components work together to implement the following parameter-responsive centrifugation process: 1) Adaptive calculation of centrifugal force: ; In the formula, It is centrifugal force; The real-time sedimentation rate of cells is identified by a microscope camera; The baseline settlement rate; Centrifugal force as reference; centrifugal force adjustment coefficient The expression used to regulate the relationship between the intensity and time of cell treatment during centrifugation is: The calculation results are constrained to a safe range using clamping functions. ; 2) Adaptive calculation of centrifugation time: ; In the formula, Centrifugation time; The reference centrifugation time; Target PBMC layer thickness; The real-time thickness of the PBMC layer is identified by a color camera. Gradient interface sharpness is determined by a color camera; time adjustment factor. The calculation results are constrained to a safe range using a clamping function: ; 3) Temperature control adaptive calculation: ; In the formula, For temperature; Reference temperature; Real-time PBMC viability, obtained from microscopic camera image analysis; temperature regulation coefficient. The calculation results are constrained to a safe range using a clamping function: ; 4) Adaptive calculation of acceleration / deceleration slope: ; ; In the formula, For interface perturbation; Accelerate the slope as a baseline; The reference deceleration slope; To accelerate the slope; The deceleration slope; the slope attenuation coefficient. The calculation result enforces lower limit protection, that is: , .

3. The method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 2, characterized in that: Based on the calculated parameters of formulas 1), 2), 3), and 4) above, the servo motor performs density gradient centrifugation according to the sequence of "stepwise acceleration → constant temperature and constant force centrifugation → stepwise deceleration". During centrifugation, the density gradient medium is Ficoll-Paque Plus with a density of 1.077 g / mL, automatically forming a four-layer structure: plasma layer → PBMC layer → medium layer → red blood cell layer. After centrifugation, a sterile aspiration needle is inserted along the inner wall of the centrifuge cup, and segmented flow rate aspiration is performed according to the cell density of the aspiration segment. ; In the formula, The real-time cell density of the aspirated segment was counted using a microscope camera. The suction flow rate is denoted as .

4. The method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S3, the concentration of CD3 antibody is 1.8–2.2 μg / ml, the concentration of CD28 antibody is 1.8–2.2 μg / ml, the concentration of IL-2 is 200 U / ml, and the incubation time is 48 h.

5. The method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S4, the purity of the sorted T cells is ≥90%.

6. The method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S5, the MOI value of lentivirus transduction is 1-5, and the transduction time is 12h.

7. The method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S6, the amplification culture uses serum-free medium, the glucose concentration is maintained at 5–10 mmol / L, the lactate concentration is <15 mmol / L, and the culture time is 5–7 days.

8. The method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S8, the concentration of the cell preparation is: The repackaged volume is 10–50 ml / bag.

9. A method for preparing CAR-T cells using a fully automated cell preparation instrument according to claim 1, characterized in that: The CAR gene target is selected from at least one of CD19, CD20, BCMA, CD22, CD30, CD33, HER2, EGFRvIII or PSMA.

Images