Method for efficiently amplifying NK cells of cryopreserved mononuclear cells in vitro and application of method

Abstract

The invention discloses an NK cell in-vitro efficient amplification method of cryopreserved mononuclear cells and application of the NK cell in-vitro efficient amplification method. The method comprises the following steps: resuscitating the cryopreserved mononuclear cells; purifying and separating NK cells; preparing a culture medium by using the composition for NK cell in-vitro amplification, and then performing staged culture on the separated and purified NK cells; the composition for NK cell in-vitro amplification comprises a composition A and a composition B, wherein the composition A contains a monoclonal antibody combination; a composition B, which contains OK432; a composition C, which contains IL-7 and IL-21; and a composition D, which contains IL-2 and IL-15. The method provided by the invention is simple in operation process, low in production cost and high in repeatability and clinical applicability, key indexes such as cell purity, amplification times and killing activity are systematically evaluated, the stability and reliability of the method are ensured, an efficient and economical cell source is provided for application of NK cells in the fields of tumor immunotherapy, virus infection resistance and the like, and the method has a wide application prospect. Important scientific research values and clinical application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of cell biotechnology, and more specifically, to a method for efficiently expanding NK cells in vitro from frozen mononuclear cells and applications thereof. Background Art

[0002] Natural killer cells (NK cells), due to their MHC-free nature, can directly recognize and kill tumor cells, offering unique advantages in tumor immunotherapy. Current clinical trials based on allogeneic NK cells have demonstrated their efficacy against hematologic malignancies and some solid tumors, and have also demonstrated a protective effect against graft-versus-host disease in hematopoietic stem cell transplantation. The development of CAR-NK technology, in particular, offers new avenues for overcoming the bottlenecks in CAR-T therapy for solid tumors.

[0003] Although umbilical cord blood and peripheral blood mononuclear cells (PBMCs) are the primary sources for obtaining NK cells (both fresh and frozen), existing isolation and expansion technologies have significant limitations. Especially for frozen samples, they struggle to meet the cell quantity and quality requirements for clinical applications. Current NK cell in vitro expansion technologies face core challenges, including low proliferation efficiency, poor functional stability, and high costs. Even using commercial NK cell culture kits, technical bottlenecks such as slow cell proliferation, insufficient surface marker expression, and limited cell numbers are still encountered, significantly increasing production costs. These issues are even more pronounced for frozen samples. These technical limitations severely restrict the large-scale application of NK cells in clinical treatment. Specifically, existing expansion protocols struggle to achieve sufficient expansion times while maintaining cell viability. Furthermore, the instability of cell functional properties during culture and the high cost of culture hinder the standardized production and clinical application of NK cell therapy.

[0004] A Chinese invention patent application (CN114075546A) discloses a NK cell expansion composition and in vitro expansion culture method. The method involves isolating peripheral blood mononuclear cells, pre-treating them in vitro with a Group A culture medium containing high-concentration IL-2, and activating them in a Group B culture medium containing CD3 and CD52 monoclonal antibodies. The cells are then expanded in vitro using a Group C culture medium. While this method can achieve an expansion multiple of approximately 550 within a 15-day culture cycle, the final cell count is still insufficient to meet the large-scale production requirements for clinical treatment. Therefore, developing a novel expansion method that can achieve a higher expansion multiple in a shorter time while maintaining NK cell functional activity remains a key technical challenge that needs to be addressed in this field. Summary of the Invention

[0005] Based on this, it is necessary to address the above technical problems, and the present invention provides a method for efficiently expanding NK cells in vitro from frozen mononuclear cells and its application.

[0006] In order to solve the above technical problems, the first aspect of the present invention proposes a method for efficient in vitro expansion of NK cells from frozen mononuclear cells, which includes the following steps: recovery of frozen mononuclear cells; purification and separation of NK cells; preparing a culture medium with a composition for in vitro expansion of NK cells and then culturing the separated and purified NK cells in stages; the composition for in vitro expansion of NK cells includes composition A, which contains a monoclonal antibody combination; composition B, which contains OK432; composition C, which contains IL-7 and IL-21; and composition D, which contains IL-2 and IL-15.

[0007] Furthermore, the culture medium includes NK cell expansion medium M1 and NK cell activation medium M2, the NK cell expansion medium M1 contains 500-2000 IU / ml IL-2 and 1-10 ng / ml IL-15, and the NK cell activation medium M2 contains 500-2000 IU / ml IL-2, 5-30 ng / ml IL-7, 1-10 ng / ml IL-15 and 5-30 ng / ml IL-21.

[0008] Furthermore, the NK cell expansion medium M1 contains 1000 IU / ml IL-2 and 5 ng / ml IL-15, and the NK cell activation medium M2 contains 1000 IU / ml IL-2, 10 ng / ml IL-7, 5 ng / ml IL-15 and 10 ng / ml IL-21.

[0009] Furthermore, the composition A contains CD2 antibody, CD16 antibody and NKG2D antibody.

[0010] Furthermore, the mononuclear cells are obtained from peripheral blood or umbilical cord blood.

[0011] Furthermore, the composition A contains 3-10 μg / ml CD2 antibody, 3-10 μg / ml CD16 antibody and 7.5-25 μg / ml NKG2D antibody; the composition B contains 0.1-0.4 μg / ml OK432; the composition C contains 0.5-3 μg / ml IL-7 and 0.5-3 μg / ml IL-21; the composition D contains 0.5-2×10 6 IU IL-2 and 1~10μg / ml IL-15.

[0012] Furthermore, the cryopreserved mononuclear cells are obtained by the following method: peripheral blood or umbilical cord blood anticoagulation is separated by density gradient centrifugation to obtain mononuclear cells, and then cryopreserved by programmed cooling in a cryopreservation solution containing 10% DMSO.

[0013] Furthermore, the staged culture of NK cells includes:

[0014] (1) Using composition A to coat a T25 culture flask;

[0015] (2) Inoculate purified NK cells to a density of 0.5-2×10 6 cells / mL, and added composition B;

[0016] (3) NK cell activation medium M2 was added on days 3 and 5;

[0017] (4) Adjust the cell density using NK cell expansion medium M1 starting on day 6 or 7;

[0018] (5) On days 10 and 12, NK cell expansion medium M1 was used to increase the cell density;

[0019] (6) On day 14, the expanded NK cells were harvested.

[0020] Furthermore, the staged culture of NK cells includes:

[0021] (1) Coat a T25 culture flask with 10 ml of PBS and 100 μL to 2 mL of composition A for 3 to 6 hours;

[0022] (2) Inoculate purified NK cells to a density of 1×10 6 cells / mL, and add 100 μL to 1 mL of composition B;

[0023] (3) On day 3, NK cell activation medium M2 containing plasma was added;

[0024] (4) On day 5, NK cell activation medium M2 containing plasma was added again;

[0025] (5) From day 6 or 7, adjust the cell density to 1×10 using NK cell expansion medium M1. 6 cells / mL;

[0026] (6) On days 10 and 12, use NK cell expansion medium M1 to increase the cell density to 1.5×10 6 cells / mL;

[0027] (7) On day 14, the expanded NK cells were harvested.

[0028] The second aspect of the present invention proposes the application of the above-mentioned method for efficiently expanding NK cells in vitro from frozen mononuclear cells in the preparation of anti-tumor immune cell preparations, and the expanded NK cells are used to prepare cell therapy drugs for treating blood tumors or solid tumors.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a method for efficiently expanding NK cells in vitro from frozen mononuclear cells. This method significantly improves the expansion efficiency and functional potential of frozen-derived NK cells through a multi-signal synergistic activation strategy and a cytokine combination. The method provided by the present invention utilizes CD2 antibodies and CD16 antibodies as the first activation signal, NKG2D antibodies as the second activation signal, and OK432 as the third activation signal, and combines cytokines such as IL-2, IL-7, IL-15, and IL-21. Without relying on trophoblast cells and without the need for pre-enrichment, high-purity, high-activity NK cells can be efficiently expanded directly from frozen umbilical cord blood or peripheral blood mononuclear cells, with an amplification multiple of up to 2000 to 2500 times. The method provided by the present invention has a simple operating procedure, low production cost, high repeatability and clinical applicability. By systematically evaluating key indicators such as cell purity, amplification multiples, and cytotoxic activity, the stability and reliability of the method are ensured, providing an efficient and economical cell source for the application of NK cells in the fields of tumor immunotherapy, antiviral infection, etc., and has important scientific research value and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0032] Figure 1A This is a graph of cell growth on the 5th day of culture in Example 1;

[0033] Figure 1B This is a graph of cell growth on the 5th day of culture in Example 2;

[0034] Figure 1C This is a graph of cell growth on the 5th day of culture in Example 3;

[0035] Figure 1D This is a graph of cell growth on the 5th day of culture in Example 4;

[0036] Figure 2 The cell proliferation curves of Examples 1 to 4 and Comparative Examples 1 to 3 after 14 days of culture are shown;

[0037] Figure 3A This is the result of flow cytometry analysis of cell phenotypes after 14 days of culture in Example 1;

[0038] Figure 3B This is the result of flow cytometry analysis of cell phenotypes after 14 days of culture in Example 2;

[0039] Figure 3C Figure 3 shows the results of flow cytometry analysis of cell phenotypes after 14 days of culture.

[0040] Figure 3D Figure 4 shows the results of flow cytometry analysis of cell phenotypes after 14 days of culture.

[0041] Figure 4A This is the result of flow cytometry analysis of cell phenotypes after 14 days of culture in Comparative Example 1;

[0042] Figure 4B This is the result of flow cytometry analysis of cell phenotypes after 14 days of culture in Comparative Example 2;

[0043] Figure 4C This is the result of flow cytometry analysis of cell phenotypes after 14 days of culture in Comparative Example 3;

[0044] Figure 5 To verify the killing rate of K562 by NK cells cultured for different days and co-cultured with K562 target cells in different ratios in Example 2;

[0045] Figure 6 To verify the killing rate of Du-145 by NK cells cultured for different days and co-cultured with Du-145 target cells in different ratios in Example 3;

[0046] Figure 7 To verify the ADCC effect of NK cells on Raji after co-culture with Raji target cells in different ratios in Example 4. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0048] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0049] It should be noted that the umbilical cords used in the embodiments of the present invention were obtained with the consent of the volunteers.

[0050] Example 1

[0051] This embodiment provides a method for efficiently expanding NK cells in vitro based on fresh peripheral blood mononuclear cells, which comprises the following steps:

[0052] 1. Acquisition of fresh peripheral blood mononuclear cells (PBMC)

[0053] (1) Blood sample stratification: Add 20 ml of density gradient separation solution Ficoll-Paque into a 50 ml centrifuge tube; then slowly stack the anticoagulated peripheral blood sample along the tube wall on the upper layer of the separation solution to ensure that the interface between the two layers is clear and there is no mixing;

[0054] (2) Centrifugation and cell separation: The mixed solution after stratification was centrifuged at 800-1200 g for 25-35 minutes at 27°C, with the centrifuge speed set to the lowest setting. After centrifugation, the solution was separated into four layers from top to bottom: plasma layer, PBMC-enriched buffy coat layer, density gradient separation layer, and red blood cell layer. The cells in the buffy coat layer were carefully aspirated and washed 2-3 times with PBS to obtain fresh PBMC.

[0055] 2. Purification of NK cells from fresh peripheral blood

[0056] (1) Cell pretreatment: 2 × 10 8 Fresh PBMC cells were centrifuged at 300 g for 5 minutes, and the supernatant was removed. The cells were resuspended in 800 μL of buffer. 200 μL of NK cell biotin antibody mixture (NK Cell Isolation Kit) was added to the cell suspension, mixed thoroughly, and incubated at 2-8°C for 5 minutes.

[0057] (2) Isolation and acquisition of NK cells: 400 μL of buffer and 200 μL of NK cell microbead mixture (NK Cell Isolation Kit reagent) were added, mixed and incubated at 2-8°C for 10 minutes; finally, NK cells were separated by magnetic cell sorting technology, centrifuged at 300 g for 5 minutes and resuspended in culture medium to obtain purified fresh peripheral blood-derived NK cells; wherein the buffer was a PBS solution containing 0.5% BSA, and all operation steps were carried out at a low temperature of 2-8°C to ensure cell activity.

[0058] 3. Composition and culture medium preparation for in vitro expansion of NK cells

[0059] (1) Composition used for in vitro expansion of NK cells

[0060] Composition A: contains 3-10 μg / ml CD2 antibody, 3-10 μg / ml CD16 antibody and 7.5-25 μg / ml NKG2D antibody. Preferably, in this embodiment, composition A contains 8 μg / ml CD2 antibody, 8 μg / ml CD16 antibody and 20 μg / ml NKG2D antibody, wherein the anti-human CD2 antibody, anti-human CD16 antibody and anti-human NKG2D antibody can also be selected from conventional monoclonal antibodies such as mouse anti-human, goat anti-human and rabbit anti-human.

[0061] Composition B: contains 0.1-0.4 μg / ml OK432, preferably, in this embodiment, contains 0.2 μg / ml OK432.

[0062] Composition C: contains 0.5-3 μg / ml IL-7 and 0.5-3 μg / ml IL-21.

[0063] D composition: contains 0.5~2×10 6 IU IL-2 and 1~10μg / ml IL-15.

[0064] (2) Culture medium preparation

[0065] NK cell expansion medium M1: Take 1 ml of D composition and add it to 1 L of NK cell serum-free medium. After mixing, the final concentration is 1000 IU / ml IL-2 and 5 ng / ml IL-15. The NK cell serum-free medium is a conventional serum-free medium, such as Lonza TheraPEAK X-VIVO 15 immune cell serum-free medium, NK cell serum-free medium version 3.0 produced by Youkang Bio, ATCC brand NK cell serum-free medium (without mammalian protein components), Beso ALyS505NK-EX serum-free NK cell medium, etc.

[0066] NK Cell Activation Medium M2: Add 1 ml of Composition C to 100 ml of Expansion Medium M1 and mix thoroughly to achieve final concentrations of 1000 IU / ml IL-2, 10 ng / ml IL-7, 5 ng / ml IL-15, and 10 ng / ml IL-21. This medium is used for the initial activation of NK cells, providing a stronger activation signal.

[0067] 4. Inducing NK cell expansion in vitro, including the following steps:

[0068] (1) Culture vessel coating (Day 0): Add 10 ml of PBS and 500 μL of the A composition to a T25 cell culture flask (treated with TC), incubate at 37°C, 5% CO2 for 3 to 6 hours, and discard the supernatant to complete the coating.

[0069] (2) Cell inoculation (Day 0): Add 500 μL of composition B, 1 mL of blood substitute (inactivated autologous plasma, AB plasma or platelet lysate) and purified NK cells (final density 0.5-2×10 6 cells / mL, the preferred final density in this example is 1×10 6 cells / mL, total volume 10 mL), and cultured at 37°C with 5% CO2.

[0070] (3) First fluid replenishment (Day 3): Add 40 mL of mixed solution (containing 2 mL of plasma and 38 mL of NK cell activation medium M2).

[0071] (4) Observation and rehydration (day 5): Evaluate the status of NK cells under a microscope, such as Figure 1A As shown, 50 mL of mixed solution (containing 2.5 mL plasma + 47.5 mL NK cell activation medium M2) was added.

[0072] (5) Adjust cell density (days 6 and 7): Measure cell density, calculate expansion multiples, add plasma and NK cell expansion medium M1, and adjust the cell concentration to 1×10 6 cells / mL.

[0073] (6) Serum-free rehydration: Measure the density, calculate the expansion multiple, and add only NK cell expansion medium M1 to adjust the concentration to 1×10 6 cells / mL.

[0074] (7) Increase the density (Day 10 and Day 12): Measure the density, calculate the expansion multiple, add NK cell expansion medium M1, and adjust the concentration to 1.5×10 6 cells / mL.

[0075] (8) Cell harvest (Day 14): Determine the final density, calculate the total expansion fold, and collect NK cells for subsequent experiments or cryopreservation.

[0076] 5. Cryopreservation of NK cells

[0077] Example 2

[0078] This embodiment provides a method for efficiently expanding NK cells in vitro based on cryopreserved peripheral blood mononuclear cells, which comprises the following steps:

[0079] 1. Cryopreservation of fresh peripheral blood mononuclear cells (PBMC)

[0080] (1) Cell preparation and cryopreservation solution preparation: After counting the cells of fresh PBMC obtained in Example 1, centrifuge at 1000×g for 10 minutes to collect the cell pellet.7 The cells were resuspended in freezing medium (10% DMSO + 90% autologous plasma, or commercial serum-free cell freezing medium) to a concentration of 10 cells / mL to prepare a cell suspension.

[0081] (2) Programmed cooling and long-term storage: The cell suspension was divided into cryopreservation tubes and programmed to cool to -80°C at a cooling rate of -1°C / min using a programmed cooling device (ThermoScientific CryoMed). After the cells were stably frozen at -80°C, they were transferred to a liquid nitrogen tank for long-term storage.

[0082] 2. Purification of NK cells from frozen peripheral blood

[0083] Recovery 2×10 8 The frozen PBMC cells were then purified from NK cells according to the method in step 2 of Example 1.

[0084] 3. Composition and culture medium preparation for in vitro expansion of NK cells

[0085] (1) The composition is the same as in Example 1

[0086] (2) Culture medium preparation: The final concentrations of NK cell expansion medium M1 are 500 IU / ml IL-2 and 1 ng / ml IL-15; the final concentrations of NK cell activation medium M2 are 500 IU / ml IL-2, 5 ng / ml IL-7, 1 ng / ml IL-15, and 5 ng / ml IL-21.

[0087] 4. To induce NK cell expansion in vitro, when the culture vessel was coated (day 0), 100 μL of composition A was added, and when the cells were inoculated (day 0), 100 μL of composition B was added. Other steps were the same as step 4 of Example 1.

[0088] 5. Cryopreservation of NK cells is the same as in Example 1. Figure 1B The graph shows the cell growth on day 5 of NK cell culture.

[0089] Example 3

[0090] This example provides a method for efficiently expanding NK cells in vitro based on fresh umbilical cord blood mononuclear cells. The difference between this method and Example 1 is that:

[0091] In step 1, fresh umbilical cord blood is used instead of fresh peripheral blood to obtain mononuclear cells (CBMCs), and then NK cells are separated and purified from CBMCs;

[0092] The final concentrations of the culture medium prepared in step 3, NK cell expansion medium M1, are 2000 IU / ml IL-2 and 10 ng / ml IL-15; the final concentrations of NK cell activation medium M2 are 2000 IU / ml IL-2, 30 ng / ml IL-7, 10 ng / ml IL-15, and 30 ng / ml IL-21;

[0093] In step 4, NK cell expansion was induced in vitro. When the culture vessel was coated (day 0), the amount of composition A added was 2 ml. When the cells were inoculated (day 0), the amount of composition B added was 1 ml. The other steps were the same as step 4 of Example 1. The subsequent NK cell freezing steps were the same as Example 1. Figure 1C The graph shows the cell growth on day 5 of NK cell culture.

[0094] Example 4

[0095] This example provides a method for efficient in vitro expansion of NK cells based on cryopreserved umbilical cord blood mononuclear cells. The only difference between this method and Example 2 is that in step 1, the umbilical cord blood mononuclear cells (CBMCs) obtained in Example 2 are used instead of the fresh peripheral blood mononuclear cells (PBMCs) obtained in Example 1 for cryopreservation and subsequent recovery experiments. Figure 1D The figure shows the cell growth on the 5th day of NK cell culture.

[0096] Figure 2 The cell proliferation curves of Examples 1 to 4 after 14 days of culture are shown. During the culture process, the NK cells of Examples 1 to 4 are in good overall growth condition. After 14 days of culture, the NK cells of Examples 1 to 2 have expanded to 1500 to 2000 times, and the NK cells of Examples 3 to 4 have expanded to 2000 to 2500 times. The expansion speed is greatly improved compared with the conventional expansion method.

[0097] Comparative Example 1

[0098] This comparative example provides a method for efficient in vitro expansion of NK cells based on frozen peripheral blood mononuclear cells. The only difference between this method and Example 2 is that in step 4, NK cell expansion is induced in vitro, and composition A is not added when the culture container is coated (day 0).

[0099] Comparative Example 2

[0100] This comparative example provides a method for efficient in vitro expansion of NK cells based on frozen peripheral blood mononuclear cells. The only difference between the method and Example 2 is that in step 4, NK cell expansion is induced in vitro, and composition B is not added when the cells are inoculated (day 0).

[0101] Comparative Example 3

[0102] This comparative example provides a method for efficient in vitro expansion of NK cells based on frozen peripheral blood mononuclear cells. The only difference between the method and Example 2 is that in the in vitro NK cell expansion step, NK cell expansion medium M1 is used instead of NK cell activation medium M2.

[0103] Figure 2 The cell proliferation curves of Comparative Examples 1 to 3 after 14 days of culture are shown. During the culture process, the overall growth state of the cells in Comparative Example 1 was poor and failed to be successfully activated and expanded, indicating that composition A is crucial for the initial activation of NK cells, and its absence causes the cells to be unable to initiate the proliferation program; and although Comparative Examples 2 to 3 successfully activated NK cells and achieved 500-1000-fold expansion, their expansion rate was slow, suggesting that composition B can significantly improve the expansion efficiency, and the specific components of M2 culture medium are indispensable for rapid expansion, while M1 can only support a basic level of proliferation.

[0104] Verification Example 1 NK cell phenotype detection

[0105] Phenotypic detection was performed on the NK cells collected on the 14th day in Examples 1 to 4 and Comparative Examples 1 to 3, respectively. The specific steps are as follows:

[0106] 1. Cell preparation: Take 1×10 6 The NK cells harvested on day 14 were centrifuged at 400 g for 5 minutes and the supernatant was discarded.

[0107] 2. Antibody labeling: resuspend the cells with 1 ml PBS, centrifuge at 400 g for 5 minutes, discard the supernatant, resuspend the cells with 100 μL PBS, add 5 μL each of FITC anti-Human CD3, PE anti-Human CD56, BV510 anti-Human CD16, and APC anti-Human CD45, mix well, and incubate at room temperature for 15 minutes in the dark.

[0108] 3. Washing step: Add 1 ml of PBS, centrifuge at 400 g for 5 minutes, discard the supernatant, repeat the wash twice, and finally resuspend the cells in 200 μL of PBS;

[0109] 4. Flow cytometry: BD flow cytometer was used to detect the cell ratios of CD3-CD56+, CD56+CD16+ and CD56+CD45+. The experimental results are shown in Table 1 and Figures 3A to 3D As shown in 4A to 4C.

[0110] Table 1: NK cell phenotype detection results in cell ratios in Examples 1 to 4 and Comparative Examples 1 to 3

[0111] <![CDATA[CD3-CD56 + ]]> <![CDATA[CD56 + CD16 + ]]> <![CDATA[CD56 + CD45 + ]]> Example 1NK 98.3％ 97.8％ 98.9％ Example 2NK 97.5％ 89.6％ 95.5％ Example 3NK 98.6％ 88.1％ 99.8％ Example 4NK 96.8％ 93.4％ 96.5％ Comparative Example 1 28.9％ 8.7％ 27.4％ Comparative Example 2 78.3％ 68.0％ 74.3％ Comparative Example 3 92.8％ 87.1％ 89.3％

[0112] Verification Example 2K562-LUC Cell Killing Assay

[0113] The NK cells cultured to days 7, 11, and 14 in Examples 1 to 4 were co-cultured with K562 cells. Under the co-culture conditions, the killing effect of the NK cells on the K562 cells was detected. The specific steps were as follows:

[0114] 1. Target cell preparation: Collect K562-LUC cells (K562 cell line stably expressing luciferase) by centrifugation and discard the supernatant; resuspend the cells in RPMI 1640 complete medium and adjust the density to 1×10 5 cells / mL;

[0115] 2. Experimental grouping and cell plating: Take 100 μL K562-LUC cell suspension (1×10 4 cells / well) were added to a light-proof 96-well white plate, with three replicates per group; effector NK cells (taken from Examples 1 to 4 and cultured to days 7, 11, and 14, respectively) were added according to different effector-target ratios (E:T = 0:1, 1:1, 5:1, and 10:1); the effector cell density was adjusted to 1×10 6 cells / mL, 0 μL (control), 10 μL (1:1), 50 μL (5:1), and 100 μL (10:1) were added respectively; the final volume of each well was made up to 200 μL with NK cell serum-free medium;

[0116] 3. Co-culture: Incubate in a 37°C, 5% CO2 incubator for 24 hours;

[0117] 4. Detection: Using Bright-Glo from Promega TM Luciferase Assay System (Cat. No. E2620) was used for detection. The 96-well plate was removed from the incubator and equilibrated at room temperature for 2 to 8 minutes. 100 μL Bright-Glo was added to each well. TM Detection reagent, use a pipette to gently pipette to mix;

[0118] 5. Experimental results: Incubate at room temperature in the dark for 2 minutes to ensure that the cells are fully lysed. Use a multi-function microplate reader to detect the chemiluminescence signal (RLU value) of each well. The results are as follows: Figure 5 As shown, the NK cells induced in vitro in Examples 1 to 4 showed significant cytotoxic activity against K562 cells, and the cytotoxic efficiency increased with the increase of the effector-target ratio.

[0119] Verification Example 3: Killing assay of Du145 cells

[0120] The NK cells cultured to days 7, 11, and 14 in Examples 1 to 4 were co-cultured with K562 cells. Under the co-culture conditions, the killing effect of the NK cells on Du145 cells was detected. The specific steps were as follows:

[0121] 1. Target cell preparation: Du145-LUC cells (Du145 cell line stably expressing Luciferase) were collected by centrifugation and the supernatant was discarded. The cells were resuspended in RPMI 1640 complete medium and the density was adjusted to 1×10 5 cells / mL;

[0122] 2. Subsequent steps are the same as those in Example 2, including experimental grouping, cell plating, co-culture, and detection. The experimental results are as follows: Figure 6 As shown, the killing effect of NK cells in Examples 1 to 4 on Du145 cells shows that the in vitro induced activation and expansion of fresh or frozen NK cells of the present invention have high killing activity on Du145 cells.

[0123] Verification Example 4: ADCC Effect Detection (Raji Cell Model)

[0124] The NK cells cultured to day 14 in Examples 1 to 4 were co-cultured with Raji cells. Under the co-culture conditions, the ADCC effect of the NK cells was detected. The specific steps were as follows:

[0125] 1. Target cell preparation: Collect Raji-LUC cells (Raji cell line stably expressing Luciferase) by centrifugation and discard the supernatant; resuspend the cells in RPMI 1640 complete medium and adjust the density to 1×10 5 cells / mL;

[0126] 2. Experimental grouping: Take 100 μL Raji target cell suspension (1×10 4 Cells / well) were added to a light-proof 96-well white plate, and experimental and control groups were set up. In the experimental group (n=3 replicates), the monoclonal antibody rituximab was added to each well to a final concentration of 5 μg / mL (200 μL system) and incubated at 37°C for 30 minutes to allow antibody binding. In the control group (n=3 replicates), no antibody was added (only an equal volume of culture medium) and incubated simultaneously for 30 minutes.

[0127] 3. Effector NK cells (taken from Examples 1 to 4 and cultured to day 14) were added at different effector-target ratios (E:T = 0:1, 1:1, 2.5:1, 5:1, 10:1); the effector cell density was adjusted to 1×10 6cells / mL, add 0 μL (0:1), 10 μL (1:1), 25 μL (2.5:1), 50 μL (5:1), and 100 μL (10:1), respectively; use NK cell serum-free medium to make up the final volume of each well to 200 μL;

[0128] 3. Co-culture: Incubate in a 37°C, 5% CO2 incubator for 24 hours;

[0129] 4. Detection: Using Bright-Glo from Promega TM Luciferase Assay System (Cat. No. E2620) was used for detection. The 96-well plate was removed from the incubator and equilibrated at room temperature for 2 to 8 minutes. 100 μL Bright-Glo was added to each well. TM Detection reagent, use a pipette to gently pipette to mix;

[0130] 5. Experimental results: Incubate at room temperature in the dark for 2 minutes to ensure that the cells are fully lysed. Use a multi-function microplate reader to detect the chemiluminescence signal (RLU value) of each well. The results are as follows: Figure 7 As shown, the NK cells induced in vitro by Examples 1 to 4 exhibited significant ADCC effects on Raji cells.

[0131] Obviously, the embodiments described above are only some of the embodiments of the present application, rather than all of the embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present application specification and the accompanying drawings, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present application.

Claims

1. A method for efficiently expanding NK cells in vitro from frozen mononuclear cells, characterized in that: The following steps are involved: Resuscitation of frozen mononuclear cells; purification and separation of NK cells; preparing a culture medium using a composition for in vitro expansion of NK cells and then culturing the separated and purified NK cells in stages; The composition for in vitro expansion of NK cells includes composition A, which contains a monoclonal antibody combination; composition B, which contains OK432; composition C, which contains IL-7 and IL-21; and composition D, which contains IL-2 and IL-15.

2. The method for efficiently expanding NK cells in vitro from frozen mononuclear cells according to claim 1, characterized in that: The culture medium includes NK cell expansion medium M1 and NK cell activation medium M2, wherein the NK cell expansion medium M1 contains 500-2000 IU / ml IL-2 and 1-10 ng / ml IL-15, and the NK cell activation medium M2 contains 500-2000 IU / ml IL-2, 5-30 ng / ml IL-7, 1-10 ng / ml IL-15 and 5-30 ng / ml IL-21.

3. The method for efficiently expanding NK cells in vitro from frozen mononuclear cells according to claim 2, characterized in that: The NK cell expansion medium M1 contains 1000 IU / ml IL-2 and 5 ng / ml IL-15, and the NK cell activation medium M2 contains 1000 IU / ml IL-2, 10 ng / ml IL-7, 5 ng / ml IL-15 and 10 ng / ml IL-21.

4. The method for efficiently expanding NK cells in vitro from frozen mononuclear cells according to claim 1, characterized in that: The composition A contains CD2 antibody, CD16 antibody and NKG2D antibody.

5. The method for efficient in vitro expansion of NK cells from frozen mononuclear cells according to claim 1, characterized in that: The mononuclear cells are obtained from peripheral blood or umbilical cord blood.

6. The method for efficiently expanding NK cells in vitro from frozen mononuclear cells according to claim 4, characterized in that: The composition A contains 3-10 μg / ml CD2 antibody, 3-10 μg / ml CD16 antibody and 7.5-25 μg / ml NKG2D antibody; the composition B contains 0.1-0.4 μg / ml OK432; the composition C contains 0.5-3 μg / ml IL-7 and 0.5-3 μg / ml IL-21; the composition D contains 0.5-2×10 6 IU IL-2 and 1~10μg / ml IL-15.

7. The method for efficiently expanding NK cells in vitro from frozen mononuclear cells according to claim 1, characterized in that: The cryopreserved mononuclear cells are obtained by the following method: peripheral blood or umbilical cord blood anticoagulation is separated by density gradient centrifugation to obtain mononuclear cells, and then cryopreserved by programmed cooling using a cryopreservation solution containing 10% DMSO.

8. The method for efficiently expanding NK cells in vitro from frozen mononuclear cells according to claim 2, characterized in that: The staged culture of NK cells includes: (1) Using composition A to coat a T25 culture flask; (2) Inoculate purified NK cells to a density of 0.5-2×10 6 cells / mL, and added composition B; (3) NK cell activation medium M2 was added on days 3 and 5; (4) Adjust the cell density using NK cell expansion medium M1 starting on day 6 or 7; (5) On days 10 and 12, NK cell expansion medium M1 was used to increase the cell density; (6) On day 14, the expanded NK cells were harvested.

9. The method for efficiently expanding NK cells in vitro from frozen mononuclear cells according to claim 8, characterized in that: The staged culture of NK cells includes: (1) Coat a T25 culture flask with 10 ml of PBS and 100 μL to 2 mL of composition A for 3 to 6 hours; (2) Inoculate purified NK cells to a density of 1×10 6 cells / mL, and add 100 μL to 1 mL of composition B; (3) On day 3, NK cell activation medium M2 containing plasma was added; (4) On day 5, NK cell activation medium M2 containing plasma was added again; (5) From day 6 or 7, adjust the cell density to 1×10 using NK cell expansion medium M1. 6 cells / mL; (6) On days 10 and 12, use NK cell expansion medium M1 to increase the cell density to 1.5×10 6 cells / mL; (7) On day 14, the expanded NK cells were harvested.

10. Use of the method for efficient in vitro expansion of NK cells from cryopreserved mononuclear cells according to any one of claims 1 to 9 in the preparation of anti-tumor immune cell preparations, characterized in that: The expanded NK cells are used to prepare cell therapy drugs for treating blood tumors or solid tumors.

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