Method for isolating and culturing high purity nk cells using apheresis

Abstract

This invention discloses a method for obtaining high-purity NK cells using apheresis, belonging to the field of cell culture. The method includes the following steps: (1) obtaining peripheral blood mononuclear cells using the apheresis blood component method; (2) adding NK cell sorting reagent to the peripheral blood mononuclear cells, mixing evenly, and allowing to stand at room temperature to obtain a cell mixture; then adding the cell mixture to the upper layer of lymphocyte separation medium, centrifuging, removing the white membrane layer and above the transparent plasma, washing the harvested cell fluid to obtain enriched NK cells; (3) purifying using CD56 positively selected magnetic beads to obtain purified NK cells; (4) inoculating the purified NK cells into a culture container coated with CD16 antibody and allowing to stand. This method can obtain NK cells with a purity of over 99%, and the cells can expand hundreds of times after 17 days of in vitro culture, exhibiting strong anti-tumor function at low target ratios.

Description

Technical Field

[0001] This invention relates to the field of cell culture, and in particular to a method for isolating and culturing high-purity NK cells obtained from apheresis. Background Technology

[0002] Natural killer (NK) cells are cytotoxic lymphocytes in the innate immune system that can kill virus-infected or cancerous cells. The field of NK cell-based cancer therapy is experiencing exponential growth and has become a crucial area of ​​innovation in immunotherapy. Therefore, to obtain large quantities of high-purity and highly cytotoxic NK cells, this study developed a method for isolating high-purity NK cells, improving NK cell purity, in vitro expansion and culture efficiency, and anti-tumor function.

[0003] NK cells can be obtained from various sources, including peripheral blood (PB-NK cells), umbilical cord blood (CB-NK cells), immortalized cell lines, hematopoietic stem cells and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). Although peripheral blood and umbilical cord blood are easier to obtain, lower in cost, and safer as primary NK cell sources, they cannot provide a large number of initial NK cells. The large-scale production of GMP clinical-grade NK cells is limited. Therefore, obtaining a large number of high-purity and highly cytotoxic NK cells to improve anti-tumor function is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for isolating and culturing high-purity NK cells using apheresis to solve the problems existing in the prior art. The method uses apheresis to generate a large number of PBMCs, and then uses RosetteSep EnrichmentCocktail NK sorting reagent and Miltenyi CD56 positive magnetic beads to enrich NK cells. After culturing for 17 days, NK cells with a purity of over 99% can be obtained, and the tumor killing rate can reach over 90% even with a low target-to-cell ratio (0.5:1).

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for isolating and culturing high-purity NK cells obtained from apheresis, comprising the following steps:

[0007] (1) Peripheral blood mononuclear cells were obtained using the apheresis method;

[0008] (2) Add NK cell sorting reagent to the peripheral blood mononuclear cells, mix well, and let stand at room temperature to obtain cell mixture; then add the cell mixture to the upper layer of lymphocyte separation solution, centrifuge, remove the white membrane layer and above the transparent plasma, wash the harvested cell solution to obtain enriched NK cells;

[0009] (3) The enriched NK cells were purified using CD56 positive magnetic beads to obtain purified NK cells;

[0010] (4) Inoculate the purified NK cells into a culture container coated with CD16 antibody and incubate statically.

[0011] Preferably, in step (1), the peripheral blood mononuclear cells number 1 billion, and are diluted 3-5 times with PBS before being mixed with the NK cell sorting reagent.

[0012] Preferably, in step (2), each milliliter of diluted peripheral blood mononuclear cells contains 10 8 The volume ratio of diluted peripheral blood mononuclear cells to NK cells sorting reagent was 25:1.

[0013] Preferably, in step (2), the volume ratio of the cell mixture to the lymphocyte separation solution is 5:4; the centrifugation conditions are: centrifugation is performed using a fast rise and slow fall mode, with a rise time of 100-150s, a fall time of 1500-2000s, a rotation speed of 400-500g, and a centrifugation time of 20-30min;

[0014] The harvested cell sap was washed with physiological saline, with a volume ratio of cell sap to physiological saline of 4:15. The washing conditions were as follows: centrifugation was performed using a constant speed rise and fall mode, with both rise and fall times of 100-150s, a rotation speed of 250-350g, and a time of 5-10min.

[0015] Preferably, in step (3), the purification using CD56 anode magnetic beads includes the following steps:

[0016] S1: Resuspend the enriched NK cells with sorting buffer, filter, centrifuge, and collect the cell pellet;

[0017] S2: After resuspending the cell pellet, add CD56 cationized magnetic beads, mix well and incubate.

[0018] S3: After incubation, add sorting buffer to the incubation mixture, centrifuge, discard the supernatant, resuspend the precipitate with sorting buffer, and sort by column to obtain purified NK cells.

[0019] Preferably, in S2, the resuspension is: per 1×10 7 Each enriched NK cell was resuspended in 50-100 μL of sorting buffer.

[0020] The volume ratio of the sorting buffer to the CD56 anode magnetic beads is (50-100):20;

[0021] The incubation process is as follows: incubate at 2–8°C for 15 minutes.

[0022] Preferably, in S3, the incubation mixture contains, at a ratio of 1 × 10 7 After enrichment, each NK cell was mixed with 1-2 mL of sorting buffer; centrifugation was performed at 200-300 g for 10 min; resuspending was performed at 1 × 10⁻⁶ cells per cell. 8 Each cell was resuspended in 400-500 μL of sorting buffer.

[0023] Preferably, the sorting buffer is prepared by mixing MACS BSA stock solution and autoMACS rinsing solution at a volume ratio of 1:20.

[0024] The present invention also provides the use of NK cells obtained by the method in the preparation of drugs that enhance the anti-non-small cell lung cancer.

[0025] Preferably, when the ratio of NK cells to non-small cell lung cancer cells is 0.5:1, the tumor cell killing efficiency is above 90%.

[0026] The present invention discloses the following technical effects:

[0027] This invention employs apheresis [i.e., collecting whole blood from a donor, separating and preserving PBMCs, while retaining the remaining blood cells and components to return to the host (apheresis component method)] to generate a large number of PBMCs. Then, NK cells are enriched using a combination of RosetteSepEnrichment Cocktail NK sorting reagent and Miltenyi CD56 positively selected magnetic beads. This separation method can not only obtain NK cells with a purity of over 99%, but also has a high initial NK cell quantity. Using factor culture, NK cells can be expanded hundreds of times in vitro after 17 days of culture, exhibiting strong anti-tumor function even under low target-to-cell ratio conditions. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The purity of NK cells in batch YB3344 was determined by D0 sorting.

[0030] Figure 2 The purity of D17 NK cells in batch YB3344;

[0031] Figure 3 The proliferation curve of NK cells from batch YB3344;

[0032] Figure 4 The curve showing the change in NK cell viability in batch YB3344;

[0033] Figure 5 The in vitro tumor killing efficiency of YB3344 batch NK cells after 24 hours;

[0034] Figure 6 The purity of NK cells in batch YB3360 was determined by D0 sorting.

[0035] Figure 7 The purity of D17 cultured NK cells in batch YB3360;

[0036] Figure 8 The proliferation curve of NK cells from batch YB3360;

[0037] Figure 9 The curve showing the change in NK cell viability in batch YB3360;

[0038] Figure 10 The in vitro tumor killing efficiency of YB3360 batch NK cells after 24 hours;

[0039] Figure 11 The purity of NK cells in batch YB3381 was determined by D0 sorting.

[0040] Figure 12 Purity of D17 NK cells in batch YB3381;

[0041] Figure 13 The proliferation curve of NK cells from batch YB3381;

[0042] Figure 14 The curve showing the change in NK cell viability in batch YB3381;

[0043] Figure 15 The tumor killing efficiency of YB3381 batch NK cells in vitro over 24 hours. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] Example 1: Isolation of NK cells

[0050] In this embodiment, RosetteSep from Stemcell is used. TM Human NK Cell Enrichment Cocktall sorting reagent was used to enrich NK cells using density gradient centrifugation with a rosette method. NK cells were then further sorted using CD56 magnetic beads via MACS technology from Miltenyi Biotec GmbH, Germany. Cell density was detected using trypan blue staining, and NK cell purity was determined by flow cytometry. The specific experimental steps are as follows:

[0051] 1.1 RosetteSep TM Human NK Cell Enrichment Cocktall Sorting Reagent for NK Cell Enrichment

[0052] 1) Use the COBE Spectra machine from Terumo Bistrom, USA to extract 1 billion single-absorption PBMCs from the donor (the date of the single-absorption operation is recorded as Day 0, and the following operations are based on this time point), and dilute with PBS 5 times.

[0053] 2) Add NK cell sorting reagent (RosetteSep) to the diluted single-collection PBMCs at a volume ratio of 25:1 (diluted PBMCs to sorting reagent). TM Mix slowly and let stand at room temperature for 20 minutes. (Human NK Cell Enrichment Cocktall)

[0054] 3) Each centrifuge tube contains 20 mL of human peripheral blood lymphocyte separation solution (Tianjin Haoyang, LTS10770125). Use a pipette to add the cells treated above to the surface of the lymphocyte separation solution in the centrifuge tube, adding 25 mL to each centrifuge tube.

[0055] 4) Centrifuge, fast rise and slow fall mode (rise: 150s; fall: 1800s), 460g, 30 min, room temperature separation.

[0056] 5) Use a pipette to aspirate the clear plasma approximately 0.5 cm above the white membrane layer in the centrifuged separation tube, then continue aspirating the remaining lymphocyte layer (including the white membrane layer). During aspiration, move the pipette tip evenly downwards along the liquid surface in the direction of aspiration. Add the harvested cells to 50 mL centrifuge tubes, add 8 mL of cell culture medium to each tube, and then add 30 mL of physiological saline. Centrifuge at 300 g for 10 min (increase: 150 s; decrease: 150 s), washing once. Before centrifugation, take a sample and count the cells to obtain enriched NK cells.

[0057] 1.2 Purification of NK cells using CD56-positive magnetic beads

[0058] 1) RosetteSep TM NK cells enriched with Human NK Cell Enrichment Cocktall sorting reagent are then further sorted using the Magnetic Activated Cell Sorting (MACS) system. First, prepare 200 mL of buffer, and dilute MACSBSA Stock Solution (130-091-376) and autoMACS Rinsing Solution (130-091-222) 1:20. Prepare and use immediately.

[0059] 2) Gently pipette and resuspend the enriched NK cell pellet in 20 mL of buffer, filter through a 40 μm cell sieve, take samples and use a cell counter to detect cell number and cell viability, and centrifuge at 300g for 10 min (9 up, 9 down).

[0060] 3) After centrifugation, discard the supernatant, every 1×10 7 The enriched NK cells were resuspended in 80 μL of buffer, and 20 μL of magnetic bead reagent (CD56 MicroBeads, human) for sorting NK cells was added. The mixture was gently pipetted and incubated at 2–8 °C for 15 minutes.

[0061] 4) After incubation, every 1×10 7 After enrichment, add 1-2 mL of buffer to the NK cells, centrifuge at 300g for 10 minutes, discard the supernatant, and divide each 1×10⁶ cells into individual portions. 8 Each cell was resuspended in 500 μL of buffer in preparation for column sorting.

[0062] 5) Place the LS separation column in the magnetic field of the MACS separator and use 3 mL of buffer to pre-wash the LS separation column.

[0063] 6) Remove the LS Columns sorting column from the MACS separator and place it above a 15 mL centrifuge tube. Add 5 mL of buffer to the magnetic column. Use the plunger to quickly push out the CD56 magnetically labeled cells and collect the cell suspension, which is the purified NK cells.

[0064] Example 2: NK cell inoculation

[0065] On NK cell culture Day 0, highly purified NK cells were cultured at a rate of 2-3 × 10⁶ cells / day. 6 Cells / mL were seeded into T75 flasks coated with CD16 antibody using IMediam for NK medium, with the addition of initial activating factors [recombinant human IL-2 (1000 IU / mL), recombinant human IL-15 (30 ng / mL), recombinant human IL-12 (30 ng / mL) and recombinant human IL-21 (50 ng / mL)], and incubated at 37°C in a 5% CO2 incubator.

[0066] Example 3: Growth Examination of NK Cells

[0067] On Day 3 of NK cell culture, the growth status of NK cells was observed. At this time, the culture medium was clear with a strong refractive index. Under a 4× microscope, the number and size of colonies were moderate in one field of view, the background was relatively clean, and there was little cell debris. After the cells were thoroughly pipetted, samples were taken for counting. The cell density was adjusted to 1×10⁶ cells / mL using IMediam for NK medium containing amplification factors [recombinant human IL-2 (1000 IU / mL) and recombinant human IL-15 (30 ng / mL)]. 6 cells / mL, and sample 2×10 cells.6 The purity of NK cells was detected using flow cytometry as described in Example 4.

[0068] During cell culture, fluid was replenished every 48 hours from Day 5 to Day 17. Cell status was observed under a microscope, and samples were taken for counting. Cell density was adjusted to 1×10⁶ cells / mL using IMediam for NK medium containing amplification factors [recombinant human IL-2 (1000 IU / mL) and recombinant human IL-15 (30 ng / mL)]. 6 cells / mL. During NK cell culture, samples were taken every 48 hours to detect changes in NK cell proliferation and purity, and cell growth curves were plotted based on cell proliferation.

[0069] Example 4: Determination of NK cell flow cytometry phenotype

[0070] 5 × 10⁵ cells were harvested from NK cells on days 0, 3, 5, 7, 9, 11, 13, 15, and 17 of cell culture. 6 NK cell suspensions were incubated with anti-human CD3 and CD56 monoclonal antibodies (purchased from BD) at room temperature in the dark for 30 minutes. CD3 levels were then analyzed by flow cytometry (using a Merck GuavaeasyCyte HT system flow cytometer). - CD56 + NK cell ratio.

[0071] Example 5: NK cell growth and proliferation assay

[0072] Using the AO / PI dual fluorescence cell counting method, 1 mL samples were taken from NK cells at days 0, 3, 5, 7, 9, 11, 13, 15, and 17 of NK cell culture for counting. The counting was repeated three times, and the NK cell density per mL was obtained based on the counting results, thus yielding the total number of NK cells.

[0073] Example 6: NK cell viability assay

[0074] Using the AO / PI dual fluorescence cell method, 1 mL samples were taken from NK cells at days 0, 3, 5, 7, 9, 11, 13, 15, and 17 of NK cell culture to determine cell viability. The cell viability of NK cells at different time points was obtained by counting the samples three times.

[0075] Example 7: Determination of the killing effect of NK cells on tumor cells 1650

[0076] This experiment used the Cell Counting Kit-8 (CCK-8) assay to detect NK cell killing activity.

[0077] 1. Detection board settings

[0078] This experiment included an effector group, a target cell group (NCI-H1650), and a culture medium control group, with three replicates prepared for each group.

[0079] (1) Effector-target group: A certain number of target cell suspensions were added to all the experimental wells of a 96-well culture plate. The required number of effector cell suspensions were added to the corresponding wells to detect the ratio of different effector cells to target cells.

[0080] (2) Target cell group: Add the target cell suspension to the 96-well culture plate. The final volume must be consistent with the experimental wells containing target cells and effector cells (adjust the volume using culture medium).

[0081] (3) Culture medium control group: Add 200 µL of culture medium to a 96-well culture plate. This type of control is required to correct for the influence of the culture medium.

[0082] 2. Operating Procedures

[0083] 2.1 The cell density was prepared at 1×10⁻⁶. 5 Target cell suspensions of 200 μL per cell were added to 96-well plates, with 3 replicates per group. The plates were then pre-cultured in an incubator for 24 hours (37°C, 5% CO2).

[0084] 2.2 Effector-target group: Discard the supernatant in the target cell wells, prepare effector cell suspensions at effector-target ratios of 0.25:1, 0.5:1, and 1:1, and add 200 μL to each target cell well.

[0085] Target cell group: Discard the supernatant in the target cell wells and add 200 μL of effector cell culture medium to each well.

[0086] Place the culture plate in an incubator and incubate for 24 hours (37℃, 5% CO2).

[0087] 2.3 Discard the supernatant, add 200 μL of DPBS to each well and wash twice. After discarding the supernatant, add 10 μL of CCK-8 solution and 100 μL of effector cell culture medium to each well, and incubate the culture plate in an incubator for 2 hours (37°C, 5% CO2).

[0088] 2.4 After incubation, the absorbance at 450 nm was measured using an ELISA reader.

[0089] 2.5 Result Calculation

[0090] Calculate the cytotoxicity percentage corresponding to the ratio of each effector cell to target cell using the following formula:

[0091]

[0092] Example 8: Proliferation, purity, and killing effect of NK cells purified from single-abortion PBMCs of batch YB3344

[0093] On May 27, 2025, as described in Examples 1-7 above, NK cell sorting and proliferation experiments were conducted using apheresis. The specific experimental parameters were: NK cell seeding density of 2 × 10⁻⁶ cells / mL. 6 / mL, the batch number is LP250527B. The initial NK cell count is 5×10⁶. 7 NK cells were sampled and counted on Days 0, 3, 5, 7, 9, 11, 13, 15, and 17 to detect changes in NK cell number, viability, and purity. NK cell growth curves and cell viability change curves were plotted based on the counts. On Day 17 of NK cell culture, the in vitro cytotoxic activity of NK cells was detected using the Cell Counting Kit-8 assay. The results are shown in Table 1. Figures 1-5 As shown.

[0094] Table 1. Changes in the purity of NK cells in batch YB3344

[0095]

[0096] According to Table 1 and Figures 1-5 As can be seen, the NK cells prepared in Example 8 expanded by approximately 600-fold on day 17, with a cell viability of over 90% and a cell purity of over 99%, maintaining a high viability and high purity. The NK cells' tumor-killing effect demonstrates that the NK cells sorted and obtained using this method still exhibit a high anti-tumor effect even at a low effector-to-target ratio. Furthermore, the NK cell killing effect gradually increases with the increase of the effector-to-target ratio, reaching over 90% in vitro at an effector-to-target ratio of 0.5:1.

[0097] Example 9: Proliferation, purity, and killing effect of purified NK cells from single-abortion PBMCs of YB3360 batch.

[0098] On June 26, 2025, as described in Examples 1-7 above, NK cell sorting and proliferation experiments were conducted using apheresis. The specific experimental parameters were: NK cell seeding density of 2 × 10⁻⁶ cells / mL. 6 / mL, the batch number is LP250626A. The initial NK cell count is 5×10⁶ / mL. 7 NK cells were sampled and counted on Days 0, 3, 5, 7, 9, 11, 13, 15, and 17 to detect changes in NK cell number, viability, and purity. NK cell growth curves and cell viability change curves were plotted based on the counts. On Day 17 of NK cell culture, the in vitro cytotoxic activity of NK cells was detected using the Cell Counting Kit-8 assay. The results are shown in Table 2. Figures 6-10 As shown.

[0099] Table 2. Changes in the purity of NK cells in batch YB3360

[0100]

[0101] According to Table 2 and Figures 6-10 As can be seen, the NK cells prepared in Example 9 expanded by approximately 966 times on day 17, with a cell viability of over 90% and a cell purity of over 99%, maintaining a high viability and high purity. The NK cells' tumor-killing effect demonstrates that the NK cells sorted and obtained using this method still exhibit a high anti-tumor effect even at a low effector-to-target ratio. Furthermore, the NK cell killing effect gradually increases with the increase of the effector-to-target ratio, achieving an in vitro anti-tumor killing effect of over 90% for NK cells with an effector-to-target ratio of 0.5:1.

[0102] Example 10: Proliferation, purity, and killing effect of purified NK cells from YB3381 batch-collected PBMCs.

[0103] On July 29, 2025, as described in Examples 1-7 above, NK cell sorting and proliferation experiments were conducted using apheresis. The specific experimental parameters were: NK cell seeding density of 2 × 10⁻⁶ cells / mL. 6 / ml, the batch number is LP250729A. The initial NK cell count is 5×10⁶. 7 NK cell counts were performed on days 0, 3, 5, 7, 9, 11, 13, 15, and 17 to detect changes in NK cell number, viability, and purity. NK cell growth curves and cell viability change curves were plotted based on the counts. On day 17 of NK cell culture, the in vitro NK cell killing activity was detected using the Cell Counting Kit-8 assay. The results are shown in Table 3. Figures 11-15 As shown.

[0104] Table 3. Changes in the purity of NK cells in batch YB3381

[0105]

[0106] According to Table 3 and Figures 11-15 It can be seen that the NK cells prepared in Example 10 of the invention expanded by about 620 times on the 17th day and the cell viability was above 90% and the cell purity was above 99%, maintaining a high viability and high purity. As can be seen from the killing effect of NK cells on tumor cells, the NK cells obtained by sorting using this method still show a high anti-tumor effect at a low effect-to-target ratio, and the killing effect of NK cells gradually increases with the increase of effect-to-target ratio. The in vitro anti-tumor killing effect of NK cells with an effect-to-target ratio of 0.5:1 can reach more than 90%.

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of NK cells in the preparation of drugs that enhance the function against non-small cell lung cancer, characterized in that, When the ratio of NK cells to non-small cell lung cancer cells is 0.5:1, the tumor cell killing efficiency is over 90%. The method for obtaining NK cells includes the following steps: (1) Obtain mononuclear cells from apheresis blood using the apheresis component method; (2) Add NK cell sorting reagent to the single blood mononuclear cells and mix well. Let stand at room temperature to obtain cell mixture. Then add the cell mixture to the upper layer of lymphocyte separation solution, centrifuge, remove the white membrane layer and above the transparent plasma, wash the harvested cell solution to obtain enriched NK cells. (3) The enriched NK cells were purified using CD56 positive magnetic beads to obtain purified NK cells; (4) Inoculate the purified NK cells into a culture container coated with CD16 antibody and incubate statically; In step (1), the number of mononuclear cells in the single blood collection is 1 billion, which are diluted 3-5 times with PBS and then mixed with the NK cell sorting reagent; In step (2), each milliliter of diluted apheresis blood contains 10 mononuclear cells. 8 The volume ratio of mononuclear cells to NK cells in the diluted apheresis blood sorting reagent was 25:

1. In step (2), the volume ratio of the cell mixture to the lymphocyte separation solution is 5:4; the centrifugation conditions are: centrifugation is performed using a fast rise and slow fall mode, with a rise time of 150s, a fall time of 1800s, a rotation speed of 460g, and a centrifugation time of 30min; The harvested cell sap was washed with physiological saline, the volume ratio of cell sap to physiological saline was 4:15, and the washing conditions were: centrifugation using constant speed rise and fall mode, the rise and fall times were both 150s, the speed was 300g, and the time was 10min. In step (3), the purification using CD56 anode magnetic beads includes the following steps: S1: Resuspend the enriched NK cells with sorting buffer, filter, centrifuge, and collect the cell pellet; S2: After resuspending the cell pellet, add CD56 cationized magnetic beads, mix well and incubate. S3: After incubation, add sorting buffer to the incubation mixture, centrifuge, discard the supernatant, resuspend the precipitate with sorting buffer, and sort by column to obtain purified NK cells; In S2, the resuspension is: per 1×10 7 Each enriched NK cell was resuspended in 50-100 μL of sorting buffer. The volume ratio of the sorting buffer to the CD56 anode magnetic beads is (50-100):20; In S3, the incubation mixture is prepared at a concentration of 1 × 10 7 After enrichment, each NK cell was mixed with 1-2 mL of sorting buffer; centrifugation was performed at 200-300 g for 10 min; resuspending was performed at 1 × 10⁻⁶ cells per cell. 8 Each cell was resuspended in 400-500 μL of sorting buffer; On NK cell culture Day 0, highly purified NK cells were cultured at a rate of 2-3 × 10⁶ cells / year. 6 Cells / mL were seeded into T75 flasks coated with CD16 antibody using IMediam for NK medium. 1000 IU / mL recombinant human IL-2, 30 ng / mL recombinant human IL-15, 30 ng / mL recombinant human IL-12 and 50 ng / mL recombinant human IL-21 were added. The flasks were incubated at 37°C in a 5% CO2 incubator for 17 days. The medium was replenished every 48 hours from Day 5 to Day 17 of cell culture.

2. The application as described in claim 1, characterized in that, In S2, the incubation is performed at 2–8°C for 15 minutes.

3. The application as described in claim 1, characterized in that, The sorting buffer solution is prepared by mixing MACS BSA stock solution and autoMACS rinsing solution at a volume ratio of 1:20.

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