Efficient cord blood NK cell multiplication culture scheme separated from autologous plasma and application of efficient cord blood NK cell multiplication culture scheme

Abstract

The invention discloses an efficient cord blood NK cell multiplication culture scheme separated from autologous plasma and application of the efficient cord blood NK cell multiplication culture scheme, by optimizing culture system design and regulating and controlling technological parameters, after 19 days of multiplication culture, the NK cell multiplication multiple can reach 2000 times or above, the cell purity is improved to 95% or above, and the tumor cell killing efficiency reaches 80% or above; the scheme successfully overcomes the core pain points of slow amplification rate, insufficient cell purity, low killing activity and the like caused by dependence of cord blood NK cells on autologous plasma in the prior art, avoids the problem of unstable culture effect of autologous plasma caused by dilution of an anticoagulant, and remarkably improves the preparation efficiency and application reliability of cord blood NK cells. And the method has extremely high clinical transformation value and wide industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a highly efficient umbilical cord blood NK cell expansion and culture protocol detached from autologous plasma and its application. Background Technology

[0002] Natural killer (NK) cells are important effector cells of the innate immune system, accounting for 5% to 15% of mononuclear cells in blood and lymphatic organs. NK cells are the body's first line of defense against viral infections and malignant cells. They can directly kill cancer cells or infected cells, and indirectly improve antibody- and T-cell-mediated responses. In addition, NK cells also have immunomodulatory functions, regulating dendritic cells, macrophages, neutrophils, and antigen-specific T-cell and B-cell responses.

[0003] NK cells have a wide range of sources, including peripheral blood, umbilical cord blood, induced pluripotent stem cells (iPSCs), and cell lines such as NK92. Among these sources, umbilical cord blood, as a readily available heterologous resource, has the advantages of wide availability and no ethical issues. The immature immune cells it contains are less likely to cause graft-versus-host disease (GVHD). In addition, umbilical cord blood contains more NK precursor cells than adult peripheral blood, making it the most promising non-invasive source of therapeutic NK cells.

[0004] In recent years, researchers have used genetic engineering to simultaneously express molecules such as IL-15 and CD137L in K562 cells and use these cells to stimulate NK cell proliferation; or they have used methods such as screening CD56- cells with magnetic beads to culture NK cells. These methods are costly, have slow proliferation rates, and produce cells with insufficient viability; moreover, these methods all require the use of autologous plasma, and the anticoagulant used in umbilical cord blood is generally a high-content liquid sodium citrate, which dilutes the obtained plasma and leads to poor cell culture results. Therefore, this application proposes a highly efficient umbilical cord blood NK cell preparation method that is free from autologous plasma. Summary of the Invention

[0005] In view of this, in order to solve the above-mentioned technical problems in the field, the purpose of this invention is to provide a highly efficient umbilical cord blood NK cell expansion and culture scheme and its application.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: The first aspect of the present invention provides a method for expanding and culturing umbilical cord blood NK cells deprived of autologous plasma, the method comprising the following steps: (1) Pretreatment of NK cell expansion culture flasks: NK cell expansion culture flasks were pretreated with coating solution containing humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46 and humanized monoclonal antibody 4-1BB. (2) Isolation of mononuclear cells from umbilical cord blood: Mononuclear cells are isolated from umbilical cord blood; (3) Inoculation of umbilical cord blood mononuclear cells: The umbilical cord blood mononuclear cells were inoculated into the NK cell expansion culture flasks pretreated in step (1) using activation medium 1; (4) First replenishment: On the 3rd day after inoculation, add an activation medium 2 with the same volume as the initial volume; (5) Second replenishment: On the 6th day after inoculation, add an activation medium of the same volume as the initial volume; (6) Third replenishment: On the 7th day after inoculation, add the same volume of amplification culture medium as the previous replenishment volume; (7) 4th-8th replenishment of culture medium: On days 9, 11, 13, 15 and 17 after inoculation, the expansion culture medium was added according to the cell density on that day, so that the cell density was maintained at (0.5-10)×10 after replenishment. 6 / mL; the amplification medium is the same as the amplification medium used in the third replenishment; (8) End of culture: After culturing for 19 days, collect NK cells.

[0007] Furthermore, the concentrations of humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46, and humanized monoclonal antibody 4-1BB in the coating solution in step (1) are 5.0-20 μg / mL, 2.0-10 μg / mL, and 5.0-20 μg / mL, respectively; The activation medium 1 mentioned in step (3) is an NK cell basal medium containing IL-2, IL-15, IL-21, IL-18, and group A streptococci for injection, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, the concentration of IL-18 is 5-20 ng / mL, and the concentration of group A streptococci for injection is 0.005-0.1 KE / mL. 3%-10% platelet lysate is also added to the medium. The activation medium 2 mentioned in step (4) is an NK cell basal medium containing IL-2, IL-15, IL-21, IL-18 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, and the concentration of IL-18 is 5-20 ng / mL, and 1%-10% platelet lysate is added to the medium at the same time; The activation medium 3 mentioned in step (5) is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium at the same time. The amplification medium in step (6) is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium.

[0008] Furthermore, the concentrations of humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46, and humanized monoclonal antibody 4-1BB in the coating solution in step (1) are 10 μg / mL, 5 μg / mL, and 10 μg / mL, respectively. Optionally, the coating solution in step (1) is a DPBS solution containing humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46, and humanized monoclonal antibody 4-1BB.

[0009] Furthermore, in step (3), the concentrations of IL-2, IL-15, IL-21, IL-18, Group A Streptococcus for Injection, and platelet lysate in the activation culture medium 1 are 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, 0.01 KE / mL, and 5%, respectively. Optionally, the inoculation density in step (3) is (0.5-10)×10 6 / mL; Optionally, the inoculation density in step (3) is 1×10⁻⁶. 6 / mL.

[0010] Furthermore, the concentrations of IL-2, IL-15, IL-21, IL-18, and platelet lysate in the activation culture medium 2 described in step (4) are 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, and 3%, respectively; Optionally, the concentrations of IL-2, IL-15, and platelet lysate in the activation medium 3 described in step (5) are 500 IU / mL, 100 IU / mL, and 3%, respectively; Optionally, the concentrations of IL-2, IL-15, and platelet lysate in the amplification medium in step (6) are 500 IU / mL, 100 IU / mL, and 3%, respectively; Optionally, the inoculation density in step (7) is 1×10⁻⁶. 6 / mL.

[0011] In a specific embodiment of the present invention, the NK cell basal culture medium used in the activation culture medium 1, activation culture medium 2 and activation culture medium 3 are all NK cell basal culture medium from Miltenyi, and the NK cell basal culture medium used in the amplification culture medium is NK cell basal culture medium from Tonglihaiyuan.

[0012] In some implementation schemes, those skilled in the art can use conventional methods to isolate mononuclear cells from umbilical cord blood (i.e., umbilical cord blood mononuclear cell isolation). The core objective of this process is to efficiently enrich mononuclear cells (including NK precursor cells, lymphocytes, monocytes, etc.) in umbilical cord blood while preserving cell viability and functional integrity, and to remove impurities such as erythrocytes, granulocytes, and platelets, so as to lay the foundation for the subsequent targeted activation and efficient expansion of NK cells.

[0013] Specifically, conventional separation methods include, but are not limited to, density gradient centrifugation, erythrocyte lysis, and immunomagnetic bead negative selection. Among these, density gradient centrifugation is the most widely used conventional technique in this field due to its simplicity, high separation efficiency, and minimal cell damage. Its operation procedure includes: Umbilical cord blood pretreatment: Collect fresh umbilical cord blood (sodium citrate, EDTA, and other conventional anticoagulants can be added to prevent clotting). First, perform preliminary centrifugation at room temperature or 4°C (500-800 g, 8-15 min) to separate the upper plasma layer from the lower blood cell sediment. If the plasma does not need to be retained, the plasma layer can be discarded directly. If it is needed for later use, the plasma can be inactivated at 56°C for 30 min and then aliquoted and frozen. Gently mix the lower blood cell sediment with an equal volume of physiological saline, PBS buffer, or serum-free culture medium to dilute the blood cells and reduce interference from red blood cells during subsequent separation. Density gradient media layup and sample loading: Select a conventional density gradient separation medium (such as Ficoll-Paque, Percoll, etc.) with a density of 1.077-1.079 g / mL. Add the separation medium to the bottom of the centrifuge tube slowly at a volume ratio of 1:2 between the separation medium and the diluted blood cell sample. Then, slowly stack the diluted blood cell sample along the wall of the centrifuge tube to ensure that a clear interface is formed between the separation medium and the sample, and avoid the two liquids mixing and affecting the separation effect. Gradient centrifugation: Place the centrifuge tubes loaded with samples into a horizontal rotor centrifuge. Set the centrifugation parameters to room temperature, 700-800 g rotation speed, and 20-30 min centrifugation time. During centrifugation, use a "slow ramp-up, zero ramp-down" mode (e.g., ramp-up at level 9, ramp-down at level 0) to ensure that cells of different densities are fully separated in the gradient medium. After centrifugation, the sample will form a distinct four-layer structure: the top layer is a mixture of plasma and diluent; the middle thin white cloud-like layer is a mononuclear cell layer; the bottom layer is a density gradient separation medium layer; and the bottom layer is a sedimentation layer of erythrocytes and granulocytes. Mononuclear cell collection and washing: Carefully aspirate the middle mononuclear cell layer with a pipette and transfer it to a new centrifuge tube; add 3-5 times the volume of washing buffer (physiological saline, PBS buffer or serum-free culture medium) to the collected mononuclear cells, mix gently, and then wash and centrifuge (500-600 g, centrifugation time 8-10 min), discarding the supernatant; repeat washing 2-3 times to thoroughly remove residual separation liquid, plasma proteins and a small number of impurity cells; Cell resuspension and quality control: After washing, the cell pellet was resuspended in an appropriate amount of NK cell basal medium (such as Miltenyi NK cell basal medium). Cell concentration and viability were detected by a cell counting chamber or a fully automated cell counter to ensure that cell viability was ≥90%. At the same time, the initial proportion of NK cells in mononuclear cells could be detected by flow cytometry to assess whether the separation effect met the requirements for subsequent culture.

[0014] In a specific embodiment of the present invention, the present invention demonstrates through experiments that by using the umbilical cord blood NK cell expansion and culture protocol described in the first aspect of the present invention, the expansion and culture can reach 2000 times after 19 days, the cell purity can reach more than 95%, and the killing efficiency against tumor cells can reach more than 80%, effectively solving the problems of slow expansion, low purity, and low killing activity of umbilical cord blood NK cells dependent on autologous plasma.

[0015] A second aspect of the present invention provides a culture medium assembly for the expansion culture of umbilical cord blood NK cells deprived of autologous plasma, the culture medium assembly comprising activation medium 1, activation medium 2, activation medium 3 and expansion medium; The activation medium 1 is a basal NK cell culture medium containing IL-2, IL-15, IL-21, IL-18, and group A streptococci for injection, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, the concentration of IL-18 is 5-20 ng / mL, and the concentration of group A streptococci for injection is 0.005-0.1 KE / mL. 3%-10% platelet lysate is also added to the culture medium. The activation medium 2 is an NK cell basal medium containing IL-2, IL-15, IL-21, IL-18 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, and the concentration of IL-18 is 5-20 ng / mL, and 1%-10% platelet lysate is added to the medium at the same time; The activation medium 3 is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium. The amplification medium is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium.

[0016] Furthermore, the concentrations of IL-2, IL-15, IL-21, IL-18, Group A Streptococcus for Injection, and platelet lysate in the activation culture medium 1 were 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, 0.01 KE / mL, and 5%, respectively. The concentrations of IL-2, IL-15, IL-21, IL-18, and platelet lysate in the activation culture medium 2 were 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, and 3%, respectively. The concentrations of IL-2, IL-15, and platelet lysate in the activation medium 3 were 500 IU / mL, 100 IU / mL, and 3%, respectively. The concentrations of IL-2, IL-15, and platelet lysate in the amplification medium were 500 IU / mL, 100 IU / mL, and 3%, respectively.

[0017] A third aspect of the present invention provides umbilical cord blood NK cells prepared by the method described in the first aspect of the present invention.

[0018] The umbilical cord blood NK cells described herein are functional immune cells obtained through a multi-dimensional optimized culture system and precisely controlled process. Their core advantage lies in completely eliminating dependence on autologous plasma. Furthermore, through humanized antibody coating, multi-cytokine synergistic activation, and a phased fluid replacement strategy, synergistic enhancement of cell proliferation and function is achieved. Compared to existing umbilical cord blood NK cells prepared using autologous plasma, these cells achieve a breakthrough improvement in core performance indicators: after 19 days of in vitro culture, cell purity (CD3+) is significantly improved. - CD56 + The cell proportion consistently reaches over 95%, with some donor samples achieving a purity of up to 96.51%, far superior to the highest purity level of 94.94% achieved by traditional processes. Cell expansion can reach over 2000-fold, with a peak expansion of 7342.64-fold, significantly exceeding the maximum expansion capacity of 25.37-fold achieved by traditional autologous plasma processes, and can expand from an initial 1.5 × 10⁻⁶ cells / mL. 7 One umbilical cord blood mononuclear cell was expanded to 3 × 10⁻⁶ 10 More than one functional NK cell is available, which fully meets the cell dose requirements for a single clinical treatment.

[0019] The umbilical cord blood NK cells not only possess excellent proliferative characteristics but also exhibit potent and stable tumor-killing function and excellent phenotypic stability. Under an effector-to-target ratio of 5:1, the killing efficiency against chronic myeloid leukemia K562 cells reaches 77.92%-95%, significantly higher than the 19.11%-69.46% killing efficiency of traditional autologous plasma-cultured cells. This high killing activity stems from the high expression of multiple key activating receptors. By day 9 of culture, NKp30 receptor expression exceeds 90%, and NKp44 receptor expression reaches 30%-50%, maintaining high levels of expression even after day 19 (NKp30 ≥ 96.63%, NKp44 ≥ 92.35%, NKG2D ≥ 99.82%). Simultaneously, the CD16 receptor expression rate remains stable above 90%, ensuring the efficient execution of antibody-dependent cell-mediated cytotoxicity (ADCC). Furthermore, the cells tested negative for sterility and mycoplasma, eliminating the risk of exogenous microbial contamination. The culture process also avoided the problem of cell activity fluctuations caused by dilution of autologous plasma with anticoagulants. The cells exhibited ≥90% activity, uniform phenotype, and stable function, providing reliable quality assurance for their clinical translation and application.

[0020] The fourth aspect of the present invention provides the application of umbilical cord blood NK cells as described in the third aspect of the present invention in the preparation of antitumor drugs.

[0021] Furthermore, the tumor is a tumor that is sensitive to NK cell killing; Optionally, the tumor may be chronic myeloid leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell lung cancer, colorectal cancer, breast cancer, gastric cancer, nasopharyngeal carcinoma, endometrial adenocarcinoma, leiomyosarcoma, angiosarcoma, or undifferentiated pleomorphic sarcoma.

[0022] In some embodiments, the tumor is a tumor sensitive to NK cell killing, and is not limited to the specific tumor types listed above. Any tumor that expresses NK cell activation receptor (such as NKG2D, NKp30, NKp46, DNAM-1, etc.) ligands on the surface of tumor cells, expresses low levels or lacks MHC-I molecules, and can be recognized and eliminated by the highly active umbilical cord blood NK cells prepared in this invention through direct killing or antibody-dependent cell-mediated cytotoxicity (ADCC) is within the scope of protection of this invention.

[0023] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention innovatively provides a highly efficient and stable expansion and culture protocol for umbilical cord blood NK cells derived from autologous plasma. Through optimized culture system design and process parameter control, after 19 days of expansion culture, NK cells can be expanded more than 2000 times, with cell purity exceeding 95%, and a tumor cell killing efficiency of over 80%. This protocol successfully overcomes the core pain points of traditional umbilical cord blood NK cell technologies, such as slow expansion rate, insufficient cell purity, and low killing activity due to reliance on autologous plasma. It also avoids the instability of culture results caused by anticoagulant dilution of autologous plasma, significantly improving the preparation efficiency and application reliability of umbilical cord blood NK cells. It possesses extremely high clinical translational value and broad industrial application prospects. Attached Figure Description

[0024] Figure 1 Flow cytometry results of NK cells after isolating mononuclear cells using the process developed in this invention; Figure 2 Flow cytometry results of NK cells from umbilical cord blood after peripheral blood mononuclear cell separation; Figure 3 Flow cytometry results of umbilical cord blood NK cells cultured on days 15 / 16 using the process developed in this invention; Figure 4 Flow cytometry results of umbilical cord blood NK cells cultured on day 19 using the process developed in this invention; Figure 5 Flow cytometry results of umbilical cord blood NK cells on day 18 of peripheral blood culture; Figure 6 The umbilical cord blood NK cell amplification curve in the process developed in this invention; Figure 7: Peripheral blood NK cell amplification curve during peripheral blood processing; Figure 8 The expression of CD16 in NK cells after 19 days of culture using the process of this invention; Figure 9 The expression of CD16 in NK cells after 19 days of autologous plasma culture; Figure 10 Results of NK purity testing in each group on Day 7 of Example 4; Figure 11 Results of NK purity testing in each group on Day 9 of Example 4; Figure 12 The purity test results of each group of NK cells on Day 7 in Example 5. Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments. A comparison is made between the present invention and the culture of umbilical cord blood NK cells using peripheral blood NK cell culture technology, to facilitate understanding and implementation of the present invention by those skilled in the art, and to further recognize the advantages of the present invention. The following specific embodiments are for illustrative purposes only and should not be construed as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in the present invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in the present invention are generally performed under conventional conditions or according to the manufacturer's recommended conditions. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the present invention as described in detail in the claims.

[0026] Example 1: Expansion and culture of umbilical cord blood NK cells in this invention 1. Pretreatment of NK cell expansion culture flasks in this invention Add 6.5 mL of DPBS containing 10 μg / mL humanized CD16 antibody (Tongli Haiyuan, GMP-TL201-0500), 5 μg / mL humanized NKp46 antibody (Biolegend, 137604), and 10 μg / mL humanized 4-1BB antibody (Baipusaisi, GMP-41LH26) to a T75 culture flask and incubate overnight at 2-8°C.

[0027] 2. Isolation of umbilical cord blood mononuclear cells (UCBMCs) 2.1 Taking 100 mL of umbilical cord blood as an example, the procedure can be adjusted accordingly if the blood volume is different. Centrifuge 100 mL of umbilical cord blood at room temperature for 10 min at 750 g with a 9°C increment and 7°C decrement to separate the plasma and blood cells.

[0028] 2.2 Carefully remove the plasma from the upper layer of blood cells using a pipette, stopping the aspiration when the plasma is about 1 cm away from the upper layer of blood cells.

[0029] 2.3 Add an equal volume of sodium chloride injection solution to the plasma, mix well, and dilute the lower layer of blood cells.

[0030] 2.4 Slowly add the diluted blood cells to a 50 mL centrifuge tube containing 15 mL Ficoll. Add 30 mL of diluted blood sample to each tube, centrifuge for 30 min at 750 g, and centrifuge at 9°C and 0°C.

[0031] 2.5 Absorb white membrane cells (UCBMC) and wash twice with 45 mL of sodium chloride injection solution.

[0032] 2.6 Resuspend cells in 40 mL of NK cell basal culture medium (Mitteni), aspirate 500 μL of cell suspension for counting, and simultaneously take 1.0 × 10⁻⁶ cells. 6 The flow cytometry analysis showed that the purity of NK cells was 18.24% (cell number UC018) (as shown in the figure). Figure 1 ).

[0033] 3. UCBMC vaccination Based on the counting result from step 2.6 above, take 1.5 × 10⁻⁶. 7 Cells at 1.0 × 10 6 / mL, inoculated into the T75cm obtained in step 1 above containing IL-2 at 500 IU / mL, IL-15 at 100 IU / mL, IL-21 at 2 IU / mL, IL-18 at 10 ng / mL, group A streptococci for injection at 0.01 KE / mL, 5% platelet lysate (HPL) and 9.8 mL of NK cell basal medium (Medrin) (i.e., activation medium 1). 2 The total volume in each culture flask is 15 mL. Culture in an incubator at 37°C and 5% CO2 concentration.

[0034] 4. First fluid resuscitation On the third day of culture, 15 mL of activation medium 2 (basal medium was Miltenyi) containing IL-2 at 500 IU / mL, IL-15 at 100 IU / mL, IL-21 at 2 IU / mL, IL-18 at 10 ng / mL, and 3% platelet lysate (HPL) was added to the culture flask, for a final volume of 30 mL.

[0035] 5. Second fluid resuscitation On day 6 of culture, 15 mL of activation medium 3 (basal medium was Miltenyi) containing IL-2 of 500 IU / mL, IL-15 of 100 IU / mL and 3% platelet lysate (HPL) was added to the culture flask, for a final volume of 45 mL.

[0036] 6. Third fluid resuscitation and bottle rotation On day 7 of culture, add 45 mL of amplification medium (basal medium is Tongli Haiyuan) containing 500 IU / mL IL-2, 100 IU / mL IL-15, and 3% platelet lysate (HPL) to the culture flask, mix well, and transfer to T225cm. 2 The final volume in the culture flask is 90 mL.

[0037] 7. Fourth fluid resuscitation On day 9 of culture, the cell concentration was measured to be 1.95 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 86mL of expansion medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 3% platelet lysate (HPL), mix well, and transfer to one T225cm cell line. 2 One culture flask contains 176 mL of the culture fluid.

[0038] 8. Fifth fluid resuscitation On day 11 of culture, the cell concentration was measured to be 2.94 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 341mL of amplification medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 3% platelet lysate (HPL), mix well, and transfer to 3 T225cm cells. 2 172 mL / bottle, 3 bottles in total.

[0039] 9. Sixth fluid resuscitation On day 13 of culture, the cell concentration was measured to be 2.99 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 1027 mL of amplification medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 3% platelet lysate (HPL), mix well, and transfer to 4 culture bags, ensuring 385 mL / bag.

[0040] 10. Seventh fluid resuscitation On day 15 of culture, the cell concentration was measured to be 2.73 × 10⁻⁶. 6 / mL, the purity of NK cells as determined by flow cytometry was 77.97% (e.g., Figure 3 (According to 1.0×10) 6 At a cell density of / mL, supplement with 2669 mL of amplification medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 3% platelet lysate (HPL), ensuring 1052 mL / bag.

[0041] 11. 8th fluid resuscitation On day 17 of culture, the cell concentration was measured to be 2.25 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 5265 mL of amplification medium (basal medium is Tongli Haiyuan) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 3% platelet lysate (HPL), ensuring 1800 mL / bag, for a total of 5 bags. Transfer the remaining suspension to T225 culture flasks, 238 mL / flask, for a total of 2 flasks.

[0042] 12. Testing On day 19 of culture, the cell concentration was measured to be 1.68 × 10⁻⁶. 6 / mL, the purity of NK cells as determined by flow cytometry was 95.69% (e.g., Figure 4 The cell suspension was tested for sterility and mycoplasma, and the results showed that both were negative. At the same time, the number of expanded cells, flow cytometry analysis, and their tumor cell killing function were also performed.

[0043] This invention involves 8 fluid replenishments and culturing for 19 days, which increases the purity of NK cells from approximately 5% in the initial umbilical cord blood mononuclear cells (UCBMCs) to over 95% after 21 days of in vitro stimulation and activation by different types of cytokines, such as IL-2, IL-21, IL-18, and IL-15. The cell count can increase by up to 2000 times, and the killing effect on tumor cells K562 can reach over 80%.

[0044] Example 2: Expanding and culturing umbilical cord blood NK cells using autologous plasma, following the peripheral blood NK cell culture process. 1. Pretreatment of cell culture flasks Add 6.5 mL of DPBS containing 10 μg / mL humanized CD16 antibody (Tongli Haiyuan, GMP-TL201-0500), 5 μg / mL humanized NKp46 antibody (Biolegend, 137604), and 10 μg / mL humanized 4-1BB antibody (Baipusaisi, GMP-41LH26) to a T75 culture flask and incubate overnight at 2-8°C.

[0045] 2. Isolation of umbilical cord blood mononuclear cells (UCBMCs) 2.1 Taking 100 mL of umbilical cord blood as an example, the procedure can be adjusted accordingly if the blood volume is different. Centrifuge 100 mL of umbilical cord blood at room temperature for 10 min at 750 g with a 9°C increment and 7°C decrement to separate the plasma and blood cells.

[0046] 2.2 Carefully remove the plasma from the upper layer of blood cells using a pipette, stopping the aspiration when the plasma is about 1 cm away from the upper layer of blood cells.

[0047] 2.3 Inactivate the plasma in a 56°C water bath for 30 minutes, aliquot it, and store it at -80°C for later use.

[0048] 2.4 Add an equal volume of sodium chloride injection solution to the plasma, mix well, and dilute the lower layer of blood cells.

[0049] 2.5 Slowly add the diluted blood cells to a 50 mL centrifuge tube containing 15 mL Ficoll. Add 30 mL of diluted blood sample to each tube, centrifuge for 30 min at 750 g, and centrifuge at 9°C and 0°C.

[0050] 2.6 Absorb white membrane cells (UCBMC) and wash twice with 45 mL of sodium chloride injection solution.

[0051] 2.7 Resuspend the cells in 40 mL of NK cell basal medium (Mitteni), and count the cells by taking 500 μL of the cell suspension. Simultaneously, take 1.0 × 10⁻⁶ cells... 6 The flow cytometry analysis showed that the purity of NK cells was 12.56% (cell number UC011) (as shown in the figure). Figure 2 ).

[0052] 3. Vaccination Based on the counting result in step 2.7 above, take 1.5 × 10. 7 Cells at 1.0 × 10 6 / mL, inoculated into the T75cm obtained in step 1 containing IL-2 at 500 IU / mL, IL-15 at 100 IU / mL, IL-21 at 2 IU / mL, IL-18 at 10 ng / mL, group A streptococci for injection at 0.01 KE / mL, 5% autologous plasma and 8.7 mL of NK cell basal culture medium (Medlan) (i.e., activation medium 1). 2 The total volume in each culture flask is 15 mL. Culture in an incubator at 37°C and 5% CO2 concentration.

[0053] 4. First fluid resuscitation On the third day of culture, 15 mL of activation medium 2 (basal medium was Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 10 ng / mL IL-18 and 5% autologous plasma was added to the culture flask, for a final volume of 30 mL.

[0054] 5. Second fluid resuscitation On the 6th day of culture, 15 mL of activation medium 3 (basal medium is Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15 and 5% autologous plasma was added to the culture flask, for a final volume of 45 mL.

[0055] 6. Third fluid resuscitation and bottle rotation On day 8 of culture, the cell concentration was measured to be 1.54 × 10⁻⁶. 6 / mL, followed by 1.0×10⁻⁶ after rehydration. 6 At a cell density of / mL, add 24 mL of expansion medium (basal medium is metronidazole) containing 1000 IU / mL IL-2, 300 IU / mL IL-15, and 5% autologous plasma to the culture flask, mix well, and transfer to a T225cm cell culture chamber. 2 69 mL / bottle, 1 bottle in total.

[0056] 7. Fourth fluid resuscitation On day 10 of culture, the cell concentration was measured to be 1.33 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 23mL of expansion medium (basal medium is Miltenyi) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 5% autologous plasma, 92mL / bottle, for a total of 1 bottle.

[0057] 8. Fifth fluid resuscitation On day 12 of culture, the cell concentration was measured to be 1.9 × 10⁻⁶. 6 / mL, at 1.0×10 6At a cell density of / mL, add 83mL of expansion medium (basal medium is Miltenyi) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 5% autologous plasma, 175 mL / bottle, for a total of 1 bottle.

[0058] 9. Sixth fluid resuscitation On day 14 of culture, the cell concentration was measured to be 1.24 × 10⁻⁶. 6 / mL, at 1.0×10 6 At a cell density of / mL, add 42mL of expansion medium (basal medium is metenitol) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 5% autologous plasma, mix well, and transfer to two T225cm cells. 2 108 mL / bottle in culture flask.

[0059] 10. Seventh fluid resuscitation On day 16 of culture, the cell concentration was measured to be 1.2 × 10⁻⁶. 6 / mL, the purity of NK cells as determined by flow cytometry was 89.33% (e.g., Figure 3 (According to 1.0×10) 6 At a cell density of / mL, add 43 mL of expansion medium (basal medium is Miltenyi) containing IL-2 500 IU / mL, IL-15 100 IU / mL, and 5% autologous plasma, 129 mL / bottle, for a total of 2 bottles.

[0060] 11. Testing On day 18 of culture, the cell concentration was measured to be 8.41 × 10⁻⁶. 5 / mL, the purity of NK cells as determined by flow cytometry was 90.61% (e.g., Figure 5 The cell suspension was tested for sterility and mycoplasma, and the results showed that both were negative. At the same time, the number of expanded cells, flow cytometry analysis, and their tumor cell killing function were also performed.

[0061] Example 3: Detection of the cytotoxic function of the expanded NK cells 1. Experimental Methods (1) After staining the target cells K562 with Calcein-AM, they were seeded in 96-well plates at a density of 20,000 cells / 100 μL. (2) Add the NK cells prepared in Example 1 and Example 2 at an effector-to-target ratio of 5:1 and incubate in the dark for 4 h; (3) Use a multi-functional microplate reader to detect the OD value and calculate the killing efficiency of each group.

[0062] 2. Experimental Results (1) Multiple batches of cell cultures were performed according to the process of this invention and the peripheral blood NK cell culture process. The results showed that the expansion of umbilical cord blood NK cells in the process of this invention was more than 2000-fold, and the purity of NK cells was more than 95%. The expansion curves are shown in the figure below. Figure 6 As shown, the purity of NK cells is as follows: Figure 4 As shown in the figure. Using autologous plasma to culture umbilical cord blood NK cells, the maximum amplification was only 25-fold, and the purity of NK cells did not reach 95%. The amplification curve is shown in the figure. Figure 7 As shown, the purity of NK cells is as follows: Figure 5 As shown.

[0063] (2) The killing efficiency of the umbilical cord blood NK cells expanded by the process of the present invention against tumor cells K562 is 77.92%-95%, while the killing efficiency of the umbilical cord blood NK cells cultured with autologous plasma against tumor cells K562 is only 19.11%-69.46% (as shown in Table 1).

[0064] (3) The expression of NK cell activation receptors DNAM-1, NKG2D, NKp30, NKp44 and NKp46 at different time points was detected by flow cytometry. The results showed that the process of the present invention could achieve high expression levels of multiple activation receptors on day 9, such as NKp30 reaching more than 90% and NKp44 reaching 30%-50%, and the activation receptors could still maintain high expression levels after 19 days of culture (as shown in Table 2).

[0065] (4) After culturing for 19 days, the expression of CD16 in the umbilical cord blood NK cells cultured by the process of this invention was all above 90% (e.g., Figure 8 However, umbilical cord blood NK cells cultured using autologous plasma do not exhibit stable CD16 expression (e.g.) Figure 9 ).

[0066] Table 1. Killing efficiency of umbilical cord blood NK cells against K562 cells after 18 or 19 days of culture.

[0067] Table 2. Detection of NK-activated receptor expression levels in umbilical cord blood at different culture time points (%)

[0068] Example 4: Comparative Experiment of Activation Culture Medium 1 and Activation Culture Medium 2 in this Invention 1. Experimental Methods (1) Pretreatment of cell culture flasks in this invention: 6.5 mL of DPBS containing 10 μg / mL humanized CD16 antibody (Tongli Haiyuan, GMP-TL201-0500), 5 μg / mL humanized NKp46 antibody (Biolegend, 137604), and 10 μg / mL humanized 4-1BB antibody (Baipusaisi, GMP-41LH26) was added to a T75 culture flask and incubated overnight at 2-8℃.

[0069] (2) Isolation of umbilical cord blood mononuclear cells (UCBMCs) Taking 100 mL of umbilical cord blood as an example, the procedure can be adjusted accordingly if the blood volume is different. Centrifuge 100 mL of umbilical cord blood at room temperature for 10 minutes, at 750 g, with an elevation of 9°C and a condensation of 7°C, to separate the plasma and blood cells.

[0070] Carefully remove the plasma from the upper layer of blood cells using a pipette, stopping when it is about 1 cm away from the upper layer of blood cells.

[0071] Add an equal volume of sodium chloride injection solution to the plasma, mix well, and dilute the lower layer of blood cells.

[0072] Slowly add the diluted blood cells to a 50 mL centrifuge tube containing 15 mL of Ficoll. Add 30 mL of diluted blood sample to each tube, centrifuge for 30 min at 750 g, and centrifuge at 9°C and 0°C.

[0073] Absorb white membrane cells (UCBMC) and wash twice with 45 mL of sodium chloride injection solution.

[0074] Resuspend the cells in 40 mL of NK cell basal culture medium (Mitteni), and count the cells by taking 500 μL of the cell suspension.

[0075] (3) UCBMC vaccination Based on the counting results, the cells were divided into 3 groups. 1.5 × 10⁻⁶ cells were collected from each group. 7 Cells at 1.0 × 10 6 / mL, the first group was inoculated with IL-2 at 500 IU / mL, IL-15 at 100 IU / mL, IL-21 at 2 IU / mL, IL-18 at 10 ng / mL, group A streptococcus for injection at 0.01 KE / mL, 5% autologous plasma and 8.89 mL of NK cell basal culture medium (Medrin) obtained in the above steps to obtain T75cm 2The total volume in each culture flask was 15 mL. The second group was inoculated with the culture medium containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 5 ng / mL IL-12, 0.01 KE / mL group A streptococci for injection, 5% autologous plasma, and 8.89 mL of NK cell basal medium (Mitteni) obtained in the above steps at T75cm. 2 In culture flasks; the third group was inoculated with the T75cm culture medium obtained in the above steps, containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 0.01 KE / mL group A streptococci for injection, 5% autologous plasma, and 8.89 mL of NK cell basal medium (Mitteni). 2 The culture was carried out in a culture flask at 37°C in an incubator with a CO2 concentration of 5%.

[0076] (4) First fluid resuscitation On day 3 of culture, 15 mL of activation medium 2-1 (basal medium was Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 10 ng / mL IL-18 and 5% autologous plasma was added to the first group of culture flasks; 15 mL of activation medium 2-2 (basal medium was Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 5 ng / mL IL-12 and 5% autologous plasma was added to the second group of culture flasks; and 15 mL of activation medium 2-3 (basal medium was Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21 and 5% autologous plasma was added to the third group of culture flasks, with a final volume of 30 mL for each group.

[0077] (5) Second fluid resuscitation On the 6th day of culture, 15 mL of activation medium 3 (basal medium is Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15 and 5% autologous plasma was added to the culture flask, for a final volume of 45 mL.

[0078] (6) Third fluid resuscitation On day 7 of culture, cell viability and fold expansion were measured by counting (see Table 3). 1.0 × 10⁶ cells were collected from each group. 6 / mL of cells was used to detect NK cell purity (e.g.) Figure 10 ) 3.0 × 10⁻⁶ for each group. 7 / mL of cells, add expansion medium (basal medium is Tongli Haiyuan) containing 500 IU / mL IL-2, 100 IU / mL IL-15 and 5% autologous plasma to 30 mL.

[0079] (7) Test on day 9 The cell concentration and viability of each group were measured (see Table 3); the purity of NK cells in each group was determined by flow cytometry (e.g., ...). Figure 11 ).

[0080] 2. Experimental Results When using autologous plasma as an additive, the activation medium 1 used on Day 0, except for the addition of 0.01 KE / mL Group A Streptococcus for injection, had the same components as the activation medium 2 used for rehydration on Day 3. Based on this, this example compared the activation factors required for the activation mediums used for rehydration in the first three days. The results showed that when 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 10 ng / mL IL-18, and 0.01 KE / mL Group A Streptococcus for injection were added to the activation medium in the first three days, the amplification folds of the two donors were 5.31-fold and 2.69-fold, respectively, and the NK cell purity was 84.85% and 63.46%, respectively, with cell viability of 74.48% and 73.23%, respectively, by Day 9. When 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 5 ng / mL IL-12, and 0.01 KE / mL Group A Streptococcus for injection were added to the activation medium during the first three days, the amplification folds of the two donors on Day 9 were 5.22-fold and 1.81-fold, respectively; the NK cell purities were 84.34% and 58.58%, respectively; and the cell viability was 70.25% and 68.69%, respectively. When 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, and 0.01 KE / mL Group A Streptococcus for injection were added to the activation medium during the first three days, the amplification folds of the two donors on Day 9 were 4.20-fold and 1.0-fold, respectively, with NK cell purities of 78.46% and 45.78%, and cell viability of 72.40% and 67.93%, respectively.

[0081] It can be seen that the activation effect of adding both IL-21 and IL-18 to the activation medium used in the first three days was better than that of adding IL-21 and IL-12; while the activation effect was the worst when only IL-21 was added, without IL-18 or IL-12. Therefore, we added IL-2 at 500 IU / mL, IL-15 at 100 IU / mL, IL-21 at 2 IU / mL, IL-18 at 10 ng / mL, and 0.01 KE / mL of group A streptococci for injection to the NK cell basal medium (Medlan) as activation medium 1; and added IL-2 at 500 IU / mL, IL-15 at 100 IU / mL, IL-21 at 2 IU / mL, and IL-18 at 10 ng / mL to the NK cell basal medium (Medlan) as activation medium 2.

[0082] Table 3. Cell counts and flow cytometry results at different time points during culture.

[0083] Example 5: Study on whether group A streptococci for injection need to be added to the activation culture medium 2 in this invention. 1. Experimental Methods (1) Pretreatment of cell culture flasks in this invention: 6.5 mL of DPBS containing 10 μg / mL humanized CD16 antibody (Tongli Haiyuan, GMP-TL201-0500), 5 μg / mL humanized NKp46 antibody (Biolegend, 137604), and 10 μg / mL humanized 4-1BB antibody (Baipusaisi, GMP-41LH26) was added to a T75 culture flask and incubated overnight at 2-8℃.

[0084] (2) Isolation of umbilical cord blood mononuclear cells (UCBMCs) Taking 100 mL of umbilical cord blood as an example, the procedure can be adjusted accordingly if the blood volume is different. Centrifuge 100 mL of umbilical cord blood at room temperature for 10 minutes, at 750 g, with an elevation of 9°C and a condensation of 7°C, to separate the plasma and blood cells.

[0085] Carefully remove the plasma from the upper layer of blood cells using a pipette, stopping when it is about 1 cm away from the upper layer of blood cells.

[0086] Add an equal volume of sodium chloride injection solution to the plasma, mix well, and dilute the lower layer of blood cells.

[0087] Slowly add the diluted blood cells to a 50 mL centrifuge tube containing 15 mL of Ficoll. Add 30 mL of diluted blood sample to each tube, centrifuge for 30 min at 750 g, and centrifuge at 9°C and 0°C.

[0088] Absorb white membrane cells (UCBMC) and wash twice with 45 mL of sodium chloride injection solution.

[0089] Resuspend the cells in 40 mL of NK cell basal culture medium (Mitteni), and count the cells by taking 500 μL of the cell suspension.

[0090] (3) UCBMC vaccination Based on the counting results, take 1.5 × 10. 7 Cells at 1.0 × 10 6 / mL was inoculated into a medium containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 10 ng / mL IL-18, 0.01 KE / mL group A streptococci for injection, 5% autologous plasma, and 11.25 mL of NK cell basal medium (Medrin) obtained by the above steps. 2 Two culture flasks were inoculated with a total volume of 15 mL each. The flasks were incubated at 37°C with a CO2 concentration of 5%.

[0091] (4) First fluid resuscitation On day 3 of culture, the first group was supplemented with 15 mL of activation medium 2-1 (basal medium was Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, and 5% autologous plasma, for a final volume of 30 mL; the second group was supplemented with 15 mL of activation medium 2-2 (basal medium was Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15, 2 IU / mL IL-21, 0.01 KE / mL of group A streptococci for injection, and 5% autologous plasma, for a final volume of 30 mL.

[0092] (5) Second fluid resuscitation On the 6th day of culture, 15 mL of activation medium 3 (basal medium is Miltenyi) containing 500 IU / mL IL-2, 100 IU / mL IL-15 and 5% autologous plasma was added to the culture flask, for a final volume of 45 mL.

[0093] (6) Test on the 7th day On day 7 of culture, cell viability and fold expansion were measured by counting (see Table 4). 1.0 × 10⁶ cells were collected from each group. 6 / mL of cells was used to detect NK cell purity (e.g.) Figure 12 ).

[0094] 2. Experimental Results In our previous studies, we noted that injectable group A streptococci play a crucial role in immune activation during NK cell culture and tumor immunotherapy. They not only activate NK cells but also comprehensively mobilize dendritic cells (DCs) and T cells, producing a synergistic anti-tumor effect. Due to their powerful immunostimulatory effect, we need to pay special attention to their dosage and duration of use to reduce quality control risks in the later production of umbilical cord blood NK preparations. Therefore, we conducted a study on the addition duration of injectable group A streptococci, with the protocol designed to add them on Day 0, Day 0, and Day 3 respectively. The results showed that adding 0.01 KE / mL injectable group A streptococci only at Day 0 inoculation and culturing to Day 7 resulted in fold increases of 6.90-fold and 5.22-fold for the two donors, respectively; NK cell purity was 49.76% and 51.03%, and cell viability was 89.26% and 85.71%, respectively. When 0.01 KE / mL of Group A Streptococcus for injection was added on Day 0 and Day 3, the amplification folds of the two donors on Day 7 were 3.99-fold and 5.07-fold, respectively, and the NK cell purity was 32.87% and 41.17%, respectively, with cell viability of 87.61% and 89.6%.

[0095] It can be seen that when the addition time of Group A Streptococcus for injection is extended to the third day, the amplification capacity of the two umbilical cord blood NK cells is actually lower, and the purity of NK cells will also decrease. Therefore, we chose to add Group A Streptococcus for injection once during Day 0 inoculation, that is, add 0.01 KE / mL Group A Streptococcus for injection to activation medium 1, and do not add Group A Streptococcus for injection to activation medium 2.

[0096] Table 4. Cell expansion and flow cytometry results on day 7 of culture.

Claims

1. A method for expanding and culturing umbilical cord blood NK cells deprived of autologous plasma, characterized in that, The method includes the following steps: (1) Pretreatment of NK cell expansion culture flasks: NK cell expansion culture flasks were pretreated with coating solution containing humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46 and humanized monoclonal antibody 4-1BB. (2) Isolation of mononuclear cells from umbilical cord blood: Mononuclear cells are isolated from umbilical cord blood; (3) Inoculation of umbilical cord blood mononuclear cells: The umbilical cord blood mononuclear cells were inoculated into the NK cell expansion culture flasks pretreated in step (1) using activation medium 1; (4) First replenishment: On the 3rd day after inoculation, add an activation medium 2 with the same volume as the initial volume; (5) Second replenishment: On the 6th day after inoculation, add an activation medium of the same volume as the initial volume; (6) Third replenishment: On the 7th day after inoculation, add the same volume of amplification culture medium as the previous replenishment volume; (7) 4th-8th replenishment of culture medium: On days 9, 11, 13, 15 and 17 after inoculation, the expansion culture medium was added according to the cell density on that day, so that the cell density was maintained at (0.5-10)×10 after replenishment. 6 / mL; the amplification medium is the same as the amplification medium used in the third replenishment; (8) End of culture: After culturing for 19 days, collect NK cells.

2. The method according to claim 1, characterized in that, The concentrations of humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46, and humanized monoclonal antibody 4-1BB in the coating solution in step (1) are 5.0-20 μg / mL, 2.0-10 μg / mL, and 5.0-20 μg / mL, respectively. The activation medium 1 mentioned in step (3) is an NK cell basal medium containing IL-2, IL-15, IL-21, IL-18, and group A streptococci for injection, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, the concentration of IL-18 is 5-20 ng / mL, and the concentration of group A streptococci for injection is 0.005-0.1 KE / mL. 3%-10% platelet lysate is also added to the medium. The activation medium 2 mentioned in step (4) is an NK cell basal medium containing IL-2, IL-15, IL-21, IL-18 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, and the concentration of IL-18 is 5-20 ng / mL, and 1%-10% platelet lysate is added to the medium at the same time; The activation medium 3 mentioned in step (5) is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium at the same time. The amplification medium in step (6) is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium.

3. The method according to claim 1, characterized in that, The concentrations of humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46, and humanized monoclonal antibody 4-1BB in the coating solution in step (1) are 10 μg / mL, 5 μg / mL, and 10 μg / mL, respectively. Optionally, the coating solution in step (1) is a DPBS solution containing humanized monoclonal antibody CD16, humanized monoclonal antibody NKp46, and humanized monoclonal antibody 4-1BB.

4. The method according to claim 1, characterized in that, In step (3), the concentrations of IL-2, IL-15, IL-21, IL-18, Group A Streptococcus for Injection, and platelet lysate in the activation medium 1 are 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, 0.01 KE / mL, and 5%, respectively. Optionally, the inoculation density in step (3) is (0.5-10)×10 6 / mL; Optionally, the inoculation density in step (3) is 1×10⁻⁶. 6 / mL.

5. The method according to claim 1, characterized in that, In step (4), the concentrations of IL-2, IL-15, IL-21, IL-18, and platelet lysate in the activation medium 2 are 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, and 3%, respectively. Optionally, the concentrations of IL-2, IL-15, and platelet lysate in the activation medium 3 described in step (5) are 500 IU / mL, 100 IU / mL, and 3%, respectively; Optionally, the concentrations of IL-2, IL-15, and platelet lysate in the amplification medium in step (6) are 500 IU / mL, 100 IU / mL, and 3%, respectively; Optionally, the inoculation density in step (7) is 1×10⁻⁶. 6 / mL.

6. A culture medium composition for the expansion and culture of umbilical cord blood NK cells deprived of autologous plasma, characterized in that, The culture medium combination includes activation culture medium 1, activation culture medium 2, activation culture medium 3, and amplification culture medium; The activation medium 1 is a basal NK cell culture medium containing IL-2, IL-15, IL-21, IL-18, and group A streptococci for injection, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, the concentration of IL-18 is 5-20 ng / mL, and the concentration of group A streptococci for injection is 0.005-0.1 KE / mL. 3%-10% platelet lysate is also added to the culture medium. The activation medium 2 is an NK cell basal medium containing IL-2, IL-15, IL-21, IL-18 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, the concentration of IL-21 is 1-10 IU / mL, and the concentration of IL-18 is 5-20 ng / mL, and 1%-10% platelet lysate is added to the medium at the same time; The activation medium 3 is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium. The amplification medium is an NK cell basal medium containing IL-2, IL-15 and platelet lysate, wherein the concentration of IL-2 is 100-1000 IU / mL, the concentration of IL-15 is 50-1000 IU / mL, and 1%-10% platelet lysate is added to the medium.

7. The culture medium combination according to claim 6, characterized in that, The concentrations of IL-2, IL-15, IL-21, IL-18, group A streptococcus for injection, and platelet lysate in the activation medium 1 were 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, 0.01 KE / mL, and 5%, respectively. The concentrations of IL-2, IL-15, IL-21, IL-18, and platelet lysate in the activation culture medium 2 were 500 IU / mL, 100 IU / mL, 2 IU / mL, 10 ng / mL, and 3%, respectively. The concentrations of IL-2, IL-15, and platelet lysate in the activation medium 3 were 500 IU / mL, 100 IU / mL, and 3%, respectively. The concentrations of IL-2, IL-15, and platelet lysate in the amplification medium were 500 IU / mL, 100 IU / mL, and 3%, respectively.

8. Umbilical cord blood NK cells prepared by any one of claims 1-5.

9. The use of umbilical cord blood NK cells as described in claim 8 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is a tumor that is sensitive to NK cell killing. Optionally, the tumor may be chronic myeloid leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell lung cancer, colorectal cancer, breast cancer, gastric cancer, nasopharyngeal carcinoma, endometrial adenocarcinoma, leiomyosarcoma, angiosarcoma, or undifferentiated pleomorphic sarcoma.

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