A method for preparing ITNK cells using a fully automated cell preparation instrument

Abstract

The application discloses a method for preparing ITNK cells by using a full-automatic cell preparation instrument, and relates to the technical field of ITNK cell preparation, and comprises the following steps: S1, T cell separation; S2, reprogramming induction; S3, ITNK cell expansion; S4, washing and concentration; and S5, quality detection. The method for preparing ITNK cells by using the full-automatic cell preparation instrument efficiently separates high-purity T cells from peripheral blood or umbilical cord blood, and completes directional reprogramming induction of the T cells into ITNK cells by means of precise temperature control and automatic material control. The quality detection link comprehensively controls cell core indexes, the mode of multi-platform manual operation is completely abandoned, the pollution risk is effectively reduced, individual differences caused by manual operation are reduced, the standardization and safety of cell preparation are improved, and it is ensured that the prepared ITNK cells meet basic clinical application requirements, and the problems of low efficiency and poor product consistency in the traditional preparation method are solved.

Description

Technical Field

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

[0002] ITNK (Induced T-cell-derived NK cells) are a new type of immune cells that acquire the phenotype and function of NK cells through reprogramming T cells. They combine the high proliferative capacity of T cells with the rapid, antigen-independent killing characteristics of NK cells, and have broad application prospects in tumor immunotherapy.

[0003] However, traditional preparation processes require manual operation across multiple platforms and steps, resulting in low efficiency and long cycles. Furthermore, open-access operations increase the risk of contamination, making it difficult to meet the requirements for clinical-grade cell preparation. Moreover, there are significant differences between different operators and laboratories, leading to poor product consistency. Summary of the Invention

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

[0006] S1, T cell separation: Peripheral blood or umbilical cord blood samples are added to the separation module, and high-purity T cells are obtained by asymmetric error-proof centrifugation coupled with Helbeck array magnetic separation.

[0007] S2, Reprogramming Induction: T cells are transferred into the reaction module, and through AI-precise temperature control and automated fluid control, T cells are induced to transform into ITNK cells by gene editing or reprogramming factors.

[0008] S3, ITNK cell expansion: ITNK cell expansion is performed in the culture module under the stimulation of feeder cells and / or cytokines.

[0009] S4. Washing and Concentration: The purification module removes residual trophoblast cells, debris and metabolites, and concentrates the ITNK cells.

[0010] S5. Quality testing: Testing the purity, activity, killing function, and sterility indicators of ITNK cells;

[0011] Steps S1 to S5 are all completed in a closed and automated manner in a fully automated cell preparation instrument.

[0012] Furthermore, in step S2, reprogramming induction includes knocking out the Bcl11b gene or inhibiting its expression.

[0013] Furthermore, in step S2, the reprogramming induction includes overexpression of Notch1, IL-15, ID3, or a combination thereof.

[0014] Furthermore, in step S3, engineered trophoblast cells are used, wherein the trophoblast cells express membrane-bound IL-15, IL-21, 4-1BBL, or OX40L.

[0015] Furthermore, in step S3, the trophoblast cells are K562 cells or mesenchymal stem cells that have been inactivated by irradiation or mitomycin C.

[0016] Furthermore, in step S3, the phenotype of ITNK cells is... .

[0017] Furthermore, in step S3, the total number of cells within the effective field of view is counted through image recognition using a microscope camera. Combined with a fixed volume of culture chamber To obtain real-time cell density The formula is as follows:

[0018]

[0019] Obtained by splitting Equation 1:

[0020]

[0021] in, for Real-time density of ITNK cells at any given moment; for Real-time density of trophoblast cells; for The number of ITNK cells at any given time; for The number of trophoblast cells at any given time;

[0022] The formula for calculating the real-time co-culture ratio is obtained from Equation 2:

[0023]

[0024] in, for The real-time co-cultivation ratio is determined; the relative deviation between the real-time ratio and the optimal ratio is calculated based on Equation 3, as follows:

[0025]

[0026] in, This represents a relative deviation in proportion; The ideal dynamic ratio of trophoblast cells to ITNK cells is determined through theoretical or experimental verification.

[0027] Furthermore, if Then automatic adjustment will be triggered, if The current process will remain unchanged.

[0028] Furthermore, based on Equation 4, the precise replenishment volume is obtained by dividing the target total number of trophoblast cells - the current number of trophoblast cells by the density of the stock solution. The formula for calculating the replenishment volume of trophoblast cells is as follows:

[0029]

[0030] Based on Equation 5, it is necessary to ensure that the co-culture ratio returns to the optimal level after supplementation. ; This represents the volume of the nutrient layer suspension that needs to be replenished. This refers to the density of the nutrient layer reserve solution.

[0031] This invention provides a method for preparing ITNK cells using a fully automated cell preparation instrument, which has the following beneficial effects:

[0032] 1. This method for preparing ITNK cells using a fully automated cell preparation instrument efficiently isolates high-purity T cells from peripheral blood or umbilical cord blood. Precise temperature control and automated material control facilitate the targeted reprogramming induction of T cells into ITNK cells. The amplification stage utilizes suitable engineered feeder cells and cytokines to achieve effective ITNK cell proliferation and ensure their specific phenotype. The washing and concentration stage effectively removes various impurities from the preparation process, achieving target cell concentration. The quality control stage comprehensively monitors core cell indicators. The entire process eliminates the need for manual operation across multiple platforms, effectively reducing the risk of contamination, minimizing individual differences caused by manual operation, improving the standardization and safety of cell preparation, and ensuring that the prepared ITNK cells meet basic clinical application requirements. This method solves the problems of low efficiency and poor product consistency associated with traditional preparation methods.

[0033] 2. This method for preparing ITNK cells using a fully automated cell preparation instrument utilizes image recognition technology to accurately determine the real-time density of two cell types. This allows for the calculation of the actual co-culture ratio and comparison with the ideal ratio. Based on the deviation, the method automatically triggers the addition of feeder cells, precisely calculating the addition volume to ensure the co-culture ratio remains within the ideal range. This adapts to the dynamic ratio requirements during cell proliferation, avoiding cell proliferation limitations caused by ratio imbalances. Furthermore, the entire control process is automated, and related control data can be recorded and traced throughout the process. This further optimizes the ITNK cell amplification effect, enhances cell activity, and significantly improves batch consistency in ITNK cell preparation, making the amplification process more scientific and precise. Attached Figure Description

[0034] Figure 1 This is a detailed flowchart illustrating the steps of the present invention;

[0035] Figure 2 This is a schematic diagram of the device appearance of the present invention;

[0036] Figure 3 This is a schematic diagram of the fluid circuit of the device of the present invention;

[0037] Figure 4 This is an image of an ITNK cell on day 5 of the present invention;

[0038] Figure 5 This is an image of an ITNK cell on day 7 of this invention. Detailed Implementation

[0039] 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.

[0040] like Figures 1-3 As shown, where, Figure 3 In the diagram, 1-22 refer to control valves. This invention provides a technical solution: a method for preparing ITNK cells using a fully automated cell preparation instrument, comprising the following steps:

[0041] S1, T cell separation: Peripheral blood or umbilical cord blood samples are added to the separation module, and high-purity T cells are obtained by asymmetric error-proof centrifugation coupled with Helbeck array magnetic separation.

[0042] The separation process specifically includes the following steps:

[0043] Step 1: The Ficoll solution flows from valve 2 through peristaltic pump 1 and then through valve 16 into the centrifuge cup;

[0044] Step 2: Whole blood flows from valve 3 through peristaltic pump 1 and then through valve 16 into the centrifuge cup;

[0045] Step 3: Centrifuge the Ficoll solution and whole blood in a centrifuge cup until the PBMCs are separated;

[0046] Step 4: Extract the intermediate layer from the separation cup through valve 16 and valve 4 into the intermediate liquid bag for later use;

[0047] 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;

[0048] Step 6: The cleaning waste liquid in the centrifuge cup is discharged from valve 16 through pump 1 and then through valve 10.

[0049] Step 7: The spare intermediate layer liquid flows from valve 4 through pump 1 and then through valve 16 into the centrifuge cup;

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Step 11: The resuspension solution flows from valve 6 through pump 1, through valve 17, and into the centrifuge cup to resuspend the cells;

[0054] S2, Reprogramming Induction: T cells are transferred into the reaction module, and through AI-controlled precise temperature and automated fluid control, T cells are induced to transform into ITNK cells by gene editing (such as CRISPR / Cas9 knockout of Bcl11b) or reprogramming factors (such as Notch1, IL-15, ID3).

[0055] Reprogramming induction includes knocking out the Bcl11b gene or inhibiting its expression; reprogramming induction also includes overexpression of Notch1, IL-15, ID3, or combinations thereof.

[0056] The process specifically includes the following steps:

[0057] Step 1: The induction reagent flows from valve 5 through pump 1 and then through valve 16 into the centrifuge cup;

[0058] Step 2: Incubate at a constant temperature for a certain period of time;

[0059] 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.

[0060] S3, ITNK cell expansion: ITNK cell expansion is performed in the culture module under the stimulation of feeder cells and / or cytokines.

[0061] Engineered trophoblast cells were used, wherein the trophoblast cells expressed at least one of membrane-bound IL-15, IL-21, 4-1BBL, or OX40L; the trophoblast cells were irradiated or mitomycin C-inactivated K562 cells or mesenchymal stem cells; the ITNK cell phenotype was as follows: ;

[0062] The process specifically includes the following steps:

[0063] Clean the tubing to be reused and adjust the liquid bag. The cleaning solution flows from valve 1 through peristaltic pump 1 and valve 17 into the centrifuge cup and mixes, cleaning the centrifuge cup. The cleaning waste solution in the centrifuge cup is discharged from valve 16 through pump 1 and valve 10. 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, ready for use. The cleaning waste solution in the centrifuge cup is discharged from valve 16 through pump 1 and valve 10. Replace bag 2 with culture medium.

[0064] Then perform the following operations:

[0065] 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.

[0066] 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.

[0067] Step 3: Under constant temperature of 37 degrees Celsius, carry out amplification culture by circulating the medium according to the set time nodes;

[0068] 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.

[0069] S4. Washing and Concentration: The purification module removes residual trophoblast cells, debris and metabolites, and concentrates the ITNK cells.

[0070] The process specifically includes the following steps:

[0071] Step 1: The washing solution flows from valve 1 through pump 1, through valve 17, into the centrifuge cup and mixes, thus washing the cells inside the centrifuge cup.

[0072] 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.

[0073] Step 3: Under centrifugal conditions, the supernatant is further removed by pump 1 through valve 17 and then through valve 10 to achieve concentration.

[0074] 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.

[0075] Step 5: The cells in the centrifuge cup are collected into the liquid bag by passing through valve 16, pump 1, and valve 7.

[0076] S5. Quality Inspection: Test the purity, activity, killing function and sterility of ITNK cells to ensure that the product meets the requirements for clinical application;

[0077] Steps S1 to S5 are all completed in a closed and automated manner in a fully automated cell preparation instrument.

[0078] Example: 50 mL of peripheral blood was collected, added to a separation module, and T cells were obtained by density gradient centrifugation, with a purity of approximately 90%.

[0079] The Bcl11b gene was knocked out using the CRISPR / Cas9 system, and the transfected cells were cultured in the iCellAuto reaction module for 24 hours.

[0080] The reprogrammed cells were co-cultured with irradiated and inactivated K562-mbIL15-4-1BBL feeder cells, with IL-15 (10 ng / mL) and IL-2 (100 IU / mL) added, and cultured in an iCellAuto culture module for 7 days, resulting in a cell number increase of approximately 500-fold. Figures 4-5 As shown;

[0081] The purification module removes trophoblast cell debris and concentrates ITNK cells to... ;

[0082] The test results show: Purity: 88%; Cell viability: 92%; Kill rate against K562 cells: 75% (E:T=10:1); Sterility test passed;

[0083] This embodiment demonstrates that the method of the present invention can efficiently prepare highly active ITNK cells;

[0084] In step S3, the total number of cells within the effective field of view is counted through image recognition using a microscope camera. Combined with a fixed volume of culture chamber To obtain real-time cell density The formula is as follows:

[0085]

[0086] Obtained by splitting Equation 1:

[0087]

[0088] in, for Real-time density of ITNK cells at any given moment; for Real-time density of trophoblast cells; for The number of ITNK cells at any given time; for The number of trophoblast cells at any given time;

[0089] The formula for calculating the real-time co-culture ratio is obtained from Equation 2:

[0090]

[0091] in, for The real-time co-cultivation ratio is determined; the relative deviation between the real-time ratio and the optimal ratio is calculated based on Equation 3, as follows:

[0092]

[0093] in, This represents a relative deviation in proportion; To determine the ideal dynamic ratio of trophoblast cells to ITNK cells, as verified theoretically or experimentally; if Then automatic adjustment will be triggered, if The current process will remain unchanged;

[0094] Based on Equation 4, the precise replenishment volume is obtained by dividing the target total number of trophoblast cells - the current number of trophoblast cells by the density of the stock solution. The formula for calculating the replenishment volume of trophoblast cells is as follows:

[0095]

[0096] Based on Equation 5, it is necessary to ensure that the co-culture ratio returns to the optimal level after supplementation. ; This represents the volume of the nutrient layer suspension that needs to be replenished. Density of the nutrient layer reservoir solution;

[0097] Specifically, the fluid module automatically delivers ITNK cell suspension and irradiated-inactivated K562-mbIL15-4-1BBL feeder cell suspension to the culture chamber via a high-precision peristaltic pump; the mixing module uses... Mix at low speed for 30 seconds, then maintain AI temperature control. and inject To cultivate;

[0098] The centrifuge module was switched to low-speed mixing mode. To avoid cell sedimentation, a microscope camera at the bottom of the centrifuge vessel is used to acquire cell images, and AI image algorithms are used to distinguish ITNK cells. With trophoblast cells;

[0099] Calculate according to formula 1. , Data is uploaded to the control module in real time; the control module calculates according to Formula 3. Calculate according to formula 4 ;like Then automatic adjustment will be triggered, if The current process will remain unchanged;

[0100] when When automatic adjustment is triggered, the control module calculates according to formula 5. (Volume replenishment); the fluid module aseptically extracts the corresponding volume of nutrient layer stock solution and delivers it to the culture chamber; the mixing module instantly mixes the fluid. ), to ensure uniform cell distribution;

[0101] The system then pulse-supplements IL-2 (100 IU / mL) and IL-15 (10 ng / mL) based on the newly measured cell density to avoid factor depletion caused by continuous supplementation, thus matching the optimal ratio for cell proliferation. One hour after supplementation, cell density is monitored again and recalculated. ,make sure The control module automatically stores the adjustment time, density, ratio, replenishment volume, and factor concentration, making the entire process traceable.

[0102] In summary, this method for preparing ITNK cells using a fully automated cell preparation instrument efficiently isolates high-purity T cells from peripheral blood or umbilical cord blood. Precise temperature control and automated material control facilitate the targeted reprogramming induction of T cells into ITNK cells. The amplification stage utilizes suitable engineered feeder cells and cytokines to achieve effective proliferation of ITNK cells and ensure their specific phenotype. The washing and concentration stage effectively removes various impurities during the preparation process, achieving target cell concentration. The quality control stage comprehensively monitors core cell indicators. The entire process eliminates the need for manual operation across multiple platforms, effectively reducing the risk of contamination, minimizing individual differences caused by manual operation, improving the standardization and safety of cell preparation, and ensuring that the prepared ITNK cells meet basic clinical application requirements. This method solves the problems of low efficiency and poor product consistency associated with traditional preparation methods.

[0103] Image recognition technology enables precise real-time determination of the density of two cell types, thereby calculating the actual co-culture ratio and comparing it with the ideal ratio. Based on the deviation, the addition of feeder cells can be automatically triggered and adjusted. The addition volume is determined through precise calculation to ensure that the co-culture ratio is always maintained within the ideal range, adapting to the dynamic ratio requirements during cell proliferation and avoiding cell proliferation restriction caused by ratio imbalance. At the same time, the entire adjustment process is completed automatically by the equipment, and the relevant adjustment data can be recorded and traced throughout the process. This further optimizes the expansion effect of ITNK cells, enhances cell activity, and significantly improves the batch consistency of ITNK cell preparation, making the expansion process more scientific and precise.

[0104] 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 ITNK cells using a fully automated cell preparation instrument, characterized in that: Includes the following steps: S1 and T cell separation: Peripheral blood or umbilical cord blood samples are added to the separation module, and high-purity T cells are obtained by density gradient centrifugation coupled with Helbeck array magnetic separation. The separation process specifically includes the following steps: Step 1: The Ficoll solution flows from valve (2) through pump 1 and then through valve (16) into the centrifuge cup; Step 2: Whole blood flows from valve (3) through 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 (4) via valve (16) into the intermediate liquid bag for later use; Step 5: The cleaning solution flows from valve (1) through 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 into valve (16) into the centrifuge cup; Step 8: The washing solution flows from valve (1) through pump 1 and through valve (17) into the centrifuge cup and is mixed to wash the cells in the centrifuge cup; Step 9: In centrifugal state, the supernatant after cleaning flows from valve (16) through pump 1 and then through valve (10) to remove the supernatant after cleaning. Step 10: In centrifugal state, the supernatant 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; S2, reprogramming inducement: T cells are transferred into the reaction module, and through AI-controlled precise temperature and automated fluid control, T cells are induced to transform into ITNK cells by gene editing or reprogramming factors. The process specifically includes the following steps: Step 1: The induction reagent flows from valve (5), through pump 1, through valve (16) into the centrifuge cup; Step 2: Incubate at a constant temperature for a certain period of time; Step 3: During the cultivation process, gas is supplied to the centrifuge cup at the set concentration through valve (18), and the gas is carbon dioxide; S3, ITNK cell expansion: In the culture module, ITNK cells are expanded under the stimulation of feeder cells and / or cytokines; S4, Washing and Concentration: The purification module removes residual trophoblast cells, debris, and metabolic products, and concentrates them to obtain ITNK cells. The process specifically includes the following steps: Step 1: The cleaning solution flows from valve (1) through pump 1, through valve (17), into the centrifuge cup and is mixed to clean the cells in the centrifuge cup; Step 2: In centrifugal state, the supernatant after cleaning flows from valve (16) through pump 1 and then through valve (10) to remove the supernatant after cleaning. Step 3: In centrifugal state, the supernatant is further removed by pump 1 from valve (17) and then flowed 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 the pump 1 from valve (16) and through valve (7); S5. Quality Inspection: To detect the purity, activity, cytotoxic function, and asepticity indicators of ITNK cells; Steps S1 to S5 are all completed in a closed and automated manner in a fully automated cell preparation instrument.

2. The method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S2, reprogramming induction includes knocking out the Bcl11b gene or inhibiting its expression.

3. The method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S2, reprogramming induction includes overexpression of Notch1, IL-15, ID3, or a combination thereof.

4. The method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S3, engineered trophoblast cells are used, which express membrane-bound IL-15, IL-21, 4-1BBL, or OX40L.

5. A method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S3, the trophoblast cells are K562 cells or mesenchymal stem cells that have been inactivated by irradiation or mitomycin C.

6. A method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S3, the ITNK cells have a CD3 phenotype. - CD56 + NKp46 + ; The process specifically includes the following steps: The reusable pipeline is cleaned and the liquid bag is adjusted. The cleaning solution flows from valve (1) through pump 1 through valve (17) into the centrifuge cup and is mixed to clean the centrifuge cup. The cleaning waste liquid in the centrifuge cup is discharged from valve (16) through pump 1 through valve (10). The cleaning solution flows from valve (1) through pump 1 through valve (17) into the centrifuge cup. The cleaning solution in the centrifuge cup flows from valve (16) through pump 1 through valve (2) to clean pipeline No. 2 and is ready for use. The cleaning waste liquid in the centrifuge cup is discharged from valve (16) through pump 1 through valve (10). The No. 2 bag is replaced with culture medium. The No. 2 pipeline is the pipeline where valve (2) and Ficoll liquid bag are located. The No. 2 bag is a Ficoll liquid bag. 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, it is necessary to change the medium: In the centrifugation state, the medium flows from valve (16) through pump 1 and through valve (10) to remove the supernatant of the culture medium. Then, the culture medium flows from valve (2) through pump 1 and 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), and the gas is carbon dioxide.

7. A method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 1, characterized in that: In step S3, the total number of cells within the effective field of view is counted through image recognition using a microscope camera. Combined with a fixed volume of culture chamber To obtain real-time cell density The formula is as follows: ； Obtained by splitting Equation 1: ； in, for Real-time density of ITNK cells at any given moment; for Real-time density of trophoblast cells; for The number of ITNK cells at any given time; for The number of trophoblast cells at any given time; The formula for calculating the real-time co-culture ratio is obtained from Equation 2: ； in, for The real-time co-cultivation ratio is determined; the relative deviation between the real-time ratio and the optimal ratio is calculated based on Equation 3, as follows: ； in, This represents a relative deviation in proportion; The ideal dynamic ratio of trophoblast cells to ITNK cells is determined through theoretical or experimental verification.

8. A method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 7, characterized in that: like Then automatic adjustment will be triggered, if The current process will remain unchanged.

9. A method for preparing ITNK cells using a fully automated cell preparation instrument according to claim 7, characterized in that: Based on Equation 4, the precise replenishment volume is obtained by dividing the target total number of trophoblast cells - the current number of trophoblast cells by the density of the stock solution. The formula for calculating the replenishment volume of trophoblast cells is as follows: ； Based on Equation 5, it is necessary to ensure that the co-culture ratio returns to the optimal level after supplementation. ; This represents the volume of the nutrient layer suspension that needs to be replenished. This refers to the density of the nutrient layer reserve solution.

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