NK cell in-vitro amplification culture method based on multi-factor induction and application of NK cell in-vitro amplification culture method

Abstract

The invention discloses an NK cell in-vitro amplification culture method based on multi-factor induction and application. In the NK cell in-vitro multiplication culture method, an IL-12, IL-15 and IL-18 cell factor combination and an IL-2 and / or IL-15 cell factor are adopted to carry out in-vitro multiplication culture on NK cells. According to the preparation method, a pure cytokine method is combined with an autologous plasma amplification means, the metabolic pathway of NK cells is changed through multi-cytokine pre-stimulation and proliferation factor continuous stimulation, differential expression of surface activated receptors and inhibitory receptors of the NK cells is caused, more inflammatory factors are released, and the NK cells have the characteristics of high proliferation and strong chemotaxis; the compound is used for activating anti-tumor immunity and shows stronger anti-tumor characteristics. The method has the characteristics of large amplification quantity, high differentiation purity and strong killing ability, also has the advantages of low cost and simplicity and convenience in operation, is safe, stable and efficient, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for in vitro expansion and culture of NK cells and its application. Background Art

[0002] Natural killer (NK) cells are a type of lymphocytes with immune surveillance and immune defense functions in the natural immune system. They can directly kill virus-infected cells and tumor cells and have immune regulatory capabilities. Different from T cells, NK cells directly recognize targets without relying on signal transduction from antigen-presenting cells (APCs), and then secrete perforin, granzyme, and cytokines to exert their functions. In addition, NK cells in the tumor microenvironment can also produce cytokines and chemokines to recruit dendritic cells (DCs) and promote the maturation of DCs to enhance the adaptive immune response. It has been confirmed that the infiltration of NK cells in in-situ tumors can effectively inhibit tumor growth; the infiltration of NK cells in highly metastatic organs can effectively maintain tumor cell dormancy and inhibit tumor metastasis. Therefore, adoptive NK cell therapy has gradually developed into a new biological immunotherapy method and is increasingly used in the treatment of solid tumors and hematological malignancies.

[0003] However, currently, the application of adoptive NK cell therapy for treating tumors, especially for treating solid tumors, still faces the following two challenges: 1. How to expand a limited number of NK cells in vitro to a sufficient amount of therapeutic NK cells with strong tumor-killing ability; 2. How to improve the ability of the infused NK cells to quickly target the tumor site and infiltrate into the solid tumor to exert anti-tumor effects. Researchers combined chimeric antigen receptor (CAR) with NK cells, namely CAR-NK, which can improve the toxicity, tumor targeting, and infiltration characteristics of NK cells. However, how to efficiently expand and obtain a sufficient amount of clinical-grade vector NK cells in vitro remains to be further solved.

[0004] According to existing research reports, the methods for in vitro expansion of NK cells are diverse, all aiming to obtain a sufficient number of highly pure NK cells to maximize their killing of tumor cells. The literature shows that the current main protocols for in vitro expansion of NK cells include feeder cell stimulation method, immunomagnetic bead sorting, and pure factor amplification method, etc. Although the expansion method stimulated by feeder cells can efficiently expand NK cells, after the NK cells expanded by this method are transfused, their in vivo proliferation potential and long-term viability will decrease; at the same time, NK cells may be contaminated by other tumor cells and have a certain risk of tumorigenesis in vivo. The NK cells obtained by the immunomagnetic bead sorting expansion method have high purity, but due to the lack of interaction between other immune cells, the amplification multiple of NK cells is not high. In addition, purifying NK cells with immunomagnetic beads has a relatively high cost and requires special instruments and equipment, which is not conducive to the large-scale production and preparation of NK cells and subsequent clinical applications. The method of stimulating and expanding NK cells with pure cytokines has good safety and high clinical application value, and is currently the mainstream method for in vitro expansion of NK cells. However, the current single pure factor amplification method for NK cells in the market still has the following deficiencies: most of them have a slow proliferation rate, low amplification efficiency, and low purity of NK cells (30% - 70%); at the same time, most of them require pre-coating with activation factors, and this process needs to be incubated in a refrigerator or a carbon dioxide incubator for several hours, increasing the complexity of the operation and the risk of bacterial contamination. Therefore, it is very necessary to develop an efficient NK cell culture method with low cost, simple preparation, high proliferation, strong chemotaxis, strong targeted tumor-killing ability and capable of large-scale in vitro amplification production, which has great clinical transformation and application value.

[0005] Patent CN106085958A proposes a method for preparing NK cells, which uses cytokines to promote the activation and proliferation ability of cells, thereby increasing the amplification multiple of NK cells and enhancing cell activity; however, the result analysis shows that the highest cell proliferation multiple can only reach 150 - 160 times after 20 days of culture, and the purity of NK cells reaches 90%; its proliferation increment and differentiation purity still need to be further improved, and it fails to effectively enhance the chemotaxis of NK cells. Summary of the Invention

[0006] The object of the present invention is to overcome the defects and deficiencies of the prior art, and provide an application of a cytokine combination of IL-12, IL-15 and IL-18 and IL-2 and / or IL-15 cytokines in the in vitro expansion culture of NK cells; through multi-cytokine induction, efficient in vitro expansion of autologous NK cells is achieved, which has the characteristics of high proliferation, high purity, strong chemotaxis and high targeted tumor-killing ability.

[0007] Another object of the present invention is to provide an in vitro expansion culture of NK cells.

[0008] Another object of the present invention is to provide a method for in vitro expansion and culture of NK cells.

[0009] Another object of the present invention is to provide an application of NK cells obtained by the above-mentioned method for in vitro expansion and culture of NK cells in the preparation of drugs for preventing and / or treating tumors.

[0010] The above objects of the present invention are achieved by the following technical solutions:

[0011] The present invention protects the application of a combination of IL-12, IL-15 and IL-18 cytokines and IL-2 and / or IL-15 cytokines in the in vitro expansion and culture of NK cells.

[0012] The present invention protects an in vitro expansion culture of NK cells, including an activation culture and an expansion culture that are independent of each other,

[0013] The activation culture includes a cell culture medium and a first active component added to the cell culture medium, and the first active component includes IL-12, IL-15 and IL-18 cytokines;

[0014] The expansion culture includes a cell culture medium and a second active component added to the cell culture medium, and the second active component includes IL-2 and / or IL-15 cytokines.

[0015] In some embodiments, in the activation culture, the concentration of the first active component is: 10-50 ng / ml of IL-12 cytokine, 1-100 ng / ml of IL-15 cytokine, and 50-100 ng / ml of IL-18 cytokine.

[0016] In some embodiments, in the expansion culture, the concentration of IL-2 cytokine is 100-1000 IU / ml, and the concentration of IL-15 cytokine is 1-50 ng / ml. Preferably, the concentration of the IL-2 cytokine is 150-300 IU / ml.

[0017] The present invention protects a method for in vitro expansion and culture of NK cells, including the following steps: using a combination of IL-12, IL-15 and IL-18 cytokines and IL-2 and / or IL-15 cytokines to expand and culture NK cells in vitro.

[0018] In some embodiments, the method for in vitro expansion and culture of NK cells includes the following steps:

[0019] S1, collecting peripheral blood and separating to obtain peripheral blood mononuclear cells and plasma;

[0020] S2, performing heat inactivation treatment on the plasma to obtain inactivated plasma;

[0021] S3. Resuspend the peripheral blood mononuclear cells and inoculate them in an activation medium for culturing for 1 - 3 days; the activation medium includes IL-12, IL-15, and IL-18 cytokines and 8 - 12 vol% heat-inactivated plasma;

[0022] S4. Resuspend the cells obtained in step S3 in an amplification medium and perform amplification culture for 4 - 6 days; the amplification medium includes IL-2 and / or IL-15 cytokines;

[0023] S5. Continue to culture the cell suspension in step S4 for 4 - 7 days, and centrifuge to collect NK cells.

[0024] The method for preparing NK cells by in vitro amplification of the present invention is based on the pure cytokine method to induce efficient in vitro amplification of autologous NK cells. It adopts the sequential use of multiple cytokines and the pre-activation strategy, relies on autologous plasma to provide nutrition, induces the amplification of NK cells, does not require pre-coating, and does not add feeder cells; it pre-stimulates NK cells by pure cytokines of IL-12, IL-15, and IL-18, and then maintains the amplification with a serum-free complete medium containing IL-2 and / or IL-15 cytokines. Finally, a large number of NK cells with high purity and high virulence can be obtained.

[0025] In some embodiments, in step S2, the heat inactivation treatment is: treating at 50 - 60 °C for 20 - 40 min.

[0026] In some embodiments, in step S3, the cell seeding density is 2 - 4×10 6 cells / ml.

[0027] In some embodiments, in step S4, the density of the cells during the amplification process is maintained at 1 - 1.2×10 6 cells / ml.

[0028] In some embodiments, the amplification medium may further include 8 - 12 vol% heat-inactivated plasma.

[0029] The present invention protects the application of NK cells obtained by the method for in vitro amplification and culture of the NK cells in the preparation of drugs for preventing and / or treating tumors.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] The present invention provides a method for in vitro expansion and preparation of NK cells, which adopts a pure cytokine method combined with autologous plasma amplification means. NK cells are pre-stimulated and induced by pure cytokines of IL-12, IL-15, and IL-18, and then amplified and maintained with a culture medium containing IL-2 and / or IL-15 cytokines. By pre-stimulating with multiple cytokines and continuously stimulating with proliferation factors, the metabolic pathway of NK cells is changed, resulting in differential expression of surface activation receptors and inhibitory receptors, and releasing more inflammatory factors. It has the characteristics of high proliferation and strong chemotaxis, and is used to activate anti-tumor immunity, showing stronger anti-tumor characteristics. At the same time, this method relies on autologous plasma to provide nutrition, induces and amplifies NK cells, does not require pre-coating, does not add feeder cells, and has a simple operation. It has the characteristics of a large amplification quantity, high differentiation purity, and strong killing ability, and also has the advantages of low cost and simple operation. It is safe, stable, and efficient, and is suitable for large-scale popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a comparison chart of the proliferation multiples of NK cells obtained by the in vitro amplification and culture methods of each example and comparative example of the present invention.

[0033] Figure 2 It is a comparison chart of the purity of NK cells obtained by the in vitro amplification and culture methods of each example and comparative example of the present invention. Among them, the left figure is a flow cytometry analysis chart, and the right figure is a statistical chart of the left figure.

[0034] Figure 3 It is a comparison chart of the cytotoxicity of NK cells obtained by the in vitro amplification and culture methods of each example and comparative example of the present invention.

[0035] Figure 4 It is a comparison chart of the surface receptor expression of NK cells obtained by the in vitro amplification and culture methods of Example 2 and Comparative Example 1 of the present invention. Among them, the left figure is the expression of cell activation receptors, and the right figure is the expression of cell inhibitory receptors.

[0036] Figure 5 It is a comparison chart of the inflammatory factor release of NK cells obtained by the in vitro amplification and culture methods of Example 2 and Comparative Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present invention will be further described below in conjunction with the specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw material reagents used in the embodiments of the present invention are conventional raw material reagents purchased.

[0038] Example 1

[0039] An in vitro amplification and culture method for NK cells includes the following steps:

[0040] S1, Isolate peripheral blood mononuclear cells (PBMC): Collect 50 ml of peripheral blood from volunteers using a blood collection tube containing sodium heparin anticoagulant. Transfer the blood sample to a 50 ml centrifuge tube inside a biosafety cabinet. Layer the blood and lymphocyte separation medium at a ratio of 1:1. Place the centrifuge tube with the layered sample in a centrifuge and centrifuge at 700 g for 20 min at room temperature, with the acceleration and deceleration settings at 1 / 0. Aspirate the buffy coat cells into another new 50 ml centrifuge tube, add physiological saline to make up to 50 ml, and centrifuge at 600 g for 10 min at room temperature. Continue to wash twice with physiological saline, and take samples for cell counting, viability, and purity detection.

[0041] S2, Inactivate autologous plasma: Heat the plasma obtained from the above separation at 56 °C for 30 minutes, centrifuge at 1000 g for 10 minutes, and take the supernatant to obtain heat-inactivated autologous plasma, which is stored temporarily at 4 °C for later use.

[0042] S3, Inductive culture: Take 5×10 7 isolated peripheral blood mononuclear cells, resuspend them in 25 ml of activation medium (containing 10 vol% autologous heat-inactivated plasma), with an inoculation density of 2×10 6 cells / ml. Transfer them to a breathable culture flask and place it in an incubator at 37 °C and 5% CO 2 for 2 days.

[0043] The composition of the activation medium is: Corning KBM 581 serum-free lymphocyte culture medium, 10 vol% autologous heat-inactivated plasma, 10 ng / ml of IL-12 cytokine, 50 ng / ml of IL-15 cytokine, and 50 ng / ml of IL-18 cytokine.

[0044] S4, Amplification culture: On the 3rd day, wash the cells twice with Corning KBM 581 serum-free lymphocyte culture medium, and finally resuspend them in 25 ml of amplification medium (containing 10 vol% autologous heat-inactivated plasma). Continue to place it in an incubator at 37 °C and 5% CO 2 for culture. On the 5th day, add 25 ml of amplification medium (containing 10 vol% autologous heat-inactivated plasma) to the culture flask. On the 7th day, take samples for counting and add the corresponding amplification medium (containing 10% autologous heat-inactivated plasma) to the culture flask to maintain the final cell density at 1×10 6 cells / ml. After the 7th day, replenish the amplification medium (without 10 vol% autologous heat-inactivated plasma) every 2 days, and keep the cell density at 1×10 6 cells / ml. During this period, depending on the volume of the culture medium, timely replace the culture flask or culture bag with a larger volume.

[0045] The components of the amplification culture medium are as follows: Corning KBM 581 serum-free lymphocyte culture medium, 10 vol% of autologous inactivated plasma (not added after the 7th day), and 100 IU / ml of IL-2 cytokine.

[0046] S5. Harvest NK cells: According to the culture status of the cells and the required cell quantity, the cells are continuously cultured for 12 days and then harvested. Transfer the cell suspension to a centrifuge tube for centrifugation and collection, and wash it 3 times with physiological saline to obtain a large amount of highly pure and highly active NK cells.

[0047] Example 2

[0048] A method for in vitro amplification and culture of NK cells, which is different from Example 1 in that the components of the amplification culture medium in this example are: Corning KBM 581 serum-free lymphocyte culture medium, 10 vol% of autologous inactivated plasma (not added after the 7th day), and 200 IU / ml of IL-2 cytokine.

[0049] Example 3

[0050] A method for in vitro amplification and culture of NK cells, which is different from Example 1 in that the components of the amplification culture medium in this example are: Corning KBM 581 serum-free lymphocyte culture medium, 10 vol% of autologous inactivated plasma (not added after the 7th day), 200 IU / ml of IL-2 cytokine, and 1 ng / ml of IL-15 cytokine.

[0051] Comparative Example 1

[0052] A method for in vitro amplification and culture of NK cells, which is processed with a commercially available NK cell induction culture kit of brand A, and includes the following steps:

[0053] S1. Isolate peripheral blood mononuclear cells (PBMC); the steps are the same as those in Example 1;

[0054] S2. Inactivate autologous plasma; the steps are the same as those in Example 1;

[0055] S3. Resuspend the PBMC isolated in step S1 in the NK amplification culture medium, inoculate it at a density of 3×10 6 cells / ml, add an NK activator according to 1% of the inoculation volume, and add 10% of autologous heat-inactivated plasma. The kit includes the NK amplification culture medium and the NK activator, which are sourced from Shenzhen Dakewei Biotechnology Co., Ltd. (Cat#: 6813532).

[0056] S4. Starting on the 3rd day, replenish the NK expansion medium every 2 days. The addition ratio of plasma is 5 vol% before the 7th day and drops to 1 vol% starting from the 7th day. Cells can be harvested after culturing until the 12th day. Maintain the NK cell density at 0.8 - 1.0×10 6 cells / mL throughout the expansion process.

[0057] Comparative Example 2

[0058] An in vitro expansion and culture method for NK cells, which is processed using a commercially available NK cell induction culture kit of Brand B, comprising the following steps:

[0059] S1. Isolate peripheral blood mononuclear cells (PBMC); the steps are the same as those in Example 1;

[0060] S2. Inactivate autologous plasma; the steps are the same as those in Example 1;

[0061] S3. Pre-coat the culture flask with NK-A reagent and PBS, and place it at 4°C overnight or treat it at 37°C for 2 h. Remove the coating solution and wash the bottom of the culture flask twice. Inoculate based on the NK complete medium at a density of 2×10 6 cells / ml, and add NK-B reagent and 10 vol% autologous heat-inactivated plasma.

[0062] On the 3rd day, replenish the NK complete medium with a volume twice the original volume, and at the same time add NK-C reagent and 5 vol% autologous heat-inactivated plasma. On the 5th day, replenish the NK complete medium with a volume twice the original volume, and at the same time add NK-D reagent and 5 vol% autologous heat-inactivated plasma. Starting from the 7th day, adjust the cell density to 0.6 - 1×10 6 cells / ml for fluid replacement (NK complete medium), and perform fluid replacement every 2 days until cells are harvested on the 12th day. The kit includes the NK complete medium, NK-A reagent, NK-B reagent, and NK-C reagent, which are sourced from Beijing Tongli Haiyuan Biotechnology Co., Ltd. (Cat#: AS22-1).

[0063] Comparative Example 3

[0064] An in vitro expansion and culture method for NK cells, which is processed using a commercially available NK cell induction culture kit of Brand C, comprising the following steps:

[0065] S1. Isolate peripheral blood mononuclear cells (PBMC); the steps are the same as those in Example 1;

[0066] S2. Inactivate autologous plasma; the steps are the same as those in Example 1;

[0067] S3. Use the SNK-A coating reagent and PBS to pre-coat the culture flask, place it at room temperature for 40 - 60 minutes or store it overnight at 2 - 8 °C until taken out before use, and then remove the coating solution. Remove the coating solution and wash the bottom of the culture flask once. Inoculate based on the NK complete medium at a density of 1 - 1.5×10 6 cells / ml, and use NK-1 medium (added with SNK-B reagent) and add 10 vol% autologous heat-inactivated plasma.

[0068] On the 3rd - 5th day, add NK-1 medium at twice the original volume, and at the same time add 5 vol% autologous heat-inactivated plasma.

[0069] Starting from the 7th day, add NK-2 medium (containing IL-2 1000 IU / ml) at 1 - 1.5 times the original culture volume, replenish the liquid every 2 days until the cells are harvested on the 12th day.

[0070] The kit includes SNK-A coating reagent, SNK-B reagent, NK complete medium, NK-1 medium and NK-2 medium, which are sourced from Zhuhai Bioscience & Technology Co., Ltd. (Cat#: AlyS505NK).

[0071] Performance Test

[0072] 1. NK Cell Proliferation

[0073] Experimental method: According to the culture methods of Examples 1 - 3 and Comparative Examples 1 - 3, on the day when NK cell culture starts and ends, namely Day0 and Day12, collect the cells respectively. After centrifuging at 400 x g for 5 min at room temperature, resuspend them in physiological saline, take samples for counting, and then analyze the proportion of CD3- and CD56+ NK cells by flow cytometry.

[0074] The manufacturer of the cell counter is Countstar, and the model is Rigel S2. The manufacturer of the flow cytometer is Beckman, and the model is CytoFLEX.

[0075] The calculation method of NK cell proliferation multiple is Day12 (proportion of NK cells × total cell amount) / Day0 (proportion of NK cells × total cell amount), and the results are as Figure 1 shown.

[0076] The results show that the NK cell culture method of the present invention has a relatively high NK cell proliferation multiple; among them, Examples 2 - 3 can obtain NK cells with a higher proliferation multiple compared to Example 1 and Comparative Examples 1 - 3; when the concentration of IL-2 cytokine reaches 200 IU / ml, the NK cell proliferation effect is better.

[0077] 2. NK Cell Purity

[0078] On the day when the NK cell culture in Examples 1-3 and Comparative Examples 1-3 ended, namely Day 12, NK cells were collected. After centrifugation at 400 g for 5 min at room temperature, they were resuspended in physiological saline, and samples were taken for flow cytometry analysis.

[0079] The specific steps were as follows: 5×10 5 cells from each group were transferred to 1.5 mL EP tubes, and the cells were washed twice with 0.5 mL of flow buffer (centrifuged at 400x g for 5 min at room temperature); according to the recommended concentration in the antibody instruction manual, the flow antibodies CD3-BV510 and CD56-BB700 were added, and incubated at 4 °C for 30 min; then after washing the cells twice with 0.5 mL of flow buffer (centrifuged at 400 g for 5 min at room temperature), they were resuspended in 0.5 mL of PBS buffer for flow cytometry detection, and the obtained results were as Figure 2 shown.

[0080] The selected flow antibodies CD3-BV510 and CD56-BB700 were purchased from BD Company. The flow buffer was PBS + 2% serum albumin. The manufacturer of the flow cytometer was Beckman, and the model was CytoFLEX.

[0081] The results showed that the purity of the NK cells obtained in Examples 1-3 and Comparative Example 1 could reach over 90%, and among them, the purity of the NK cells obtained in Example 2 was the highest, reaching 96.59%.

[0082] 3. Anti-tumor effect of NK cells

[0083] The NK cells amplified in Examples 1-3 and Comparative Examples 1-3 were used as effector cells, and the human gastric cancer cell line HGC-27 was used as the target cell for tumor killing experiments.

[0084] The specific steps were as follows: In a 96-well plate, 5×10 6 NK cells and 5×10 5 tumor cells were added to each well to make the effector-to-target ratio 10:1, and then incubated in an incubator at 37 °C and 5% CO 2 for 24 h. Toxicity detection was carried out according to the instructions of the CFSE / 7-AAD double-staining cell cytotoxicity detection kit. Flow cytometry analysis was performed, and the obtained results were as Figure 3 shown. The CFSE / 7-AAD double-staining cell cytotoxicity detection kit was purchased from BD Company.

[0085] The results showed that the NK cells of the present invention had excellent killing ability against gastric cancer cells HGC-27, and among them, the NK cells amplified in Example 2 had a greater advantage in killing gastric cancer cells HGC-27.

[0086] 4. Expression of surface activation markers and inhibitory markers of NK cells

[0087] Based on the results of NK cell proliferation, purity, and tumor killing, it can be found that the NK cells obtained in Example 2 of the present invention have obvious advantages; and Comparative Example 1 also has relatively obvious advantages compared with the other two comparative examples. Next, this experiment further tested the expression of surface activation markers and inhibitory markers of the NK cells obtained in Example 2 and Comparative Example 1.

[0088] The specific steps are as follows: Collect the NK cells obtained in Example 2 and Comparative Example 1 (8×10 6 ) into 1.5 mL EP tubes. After washing the cells twice with 0.5 mL of flow buffer (centrifuging at 400x g at room temperature for 5 min), divide them into 8 tubes evenly, that is, 5×10 5 cells / tube; According to the recommended concentration in the antibody instruction manual, add the flow antibodies CD3 and CD56 (for determining the NK cell population), and then add the flow antibodies CD25, CD69, NKG2D, CD107a, CD158a, CD158b, and NKG2A respectively. The remaining 1 tube is used as a blank control, and incubate at 4°C for 30 min; then wash the cells twice with 0.5 mL of flow buffer (centrifuging at 400x g at room temperature for 5 min), and resuspend them in 0.5 mL of PBS buffer for flow cytometry detection. The obtained results are as Figure 4 shown.

[0089] The flow antibodies CD25, CD69, NKG2D, CD107a, CD158a, CD158b, and NKG2A are all purchased from BD Company.

[0090] The results show that compared with Comparative Example 1, the expression of the NK cell activation markers CD25, CD69, NKG2D, and CD107a on the surface of the NK cells obtained in Example 2 increases, while the expression of the NK cell surface inhibitory markers CD158a, CD158b, and NKG2A decreases. It shows that the NK cells obtained in Example 2 of the present invention have a higher activation level. The higher expressed CD25 is the IL2 receptor, which may be the reason for the higher proliferation multiple of the NK cells in Example 2.

[0091] 5. Release of inflammatory factors by NK cells

[0092] The release of inflammatory cytokines by NK cells can enhance the acquired immune response and contribute to recruiting and activating the anti-tumor functions of other immune cells.

[0093] In this experiment, the NK cells obtained in Example 2 and Comparative Example 1 were collected respectively (1.5×10 6) Resuspend in 0.5 mL of medium, add 6 μL of monensin, and continue to incubate in the incubator for 3.5 h to block the release of pro-inflammatory factors; transfer the cells to a 1.5 mL EP tube, wash the cells twice with 0.5 mL of flow buffer (centrifuge at 400 xg at room temperature for 5 min), then according to the flow antibody instruction manual, add CD3 and CD56 antibodies, and incubate at 4 °C for 30 min for staining to determine the NK cell population; add 250 μL of Fixation / Permeablization Kit, incubate at 4 °C in the dark for 20 min for fixation and permeabilization, after washing twice with 0.5 mL of flow buffer, divide the cells into 3 tubes, that is, 5×10 5 cells / tube; according to the recommended concentration of the antibody instruction manual, add the flow antibodies IFN-γ and TNF-α respectively, and leave 1 tube as a control, incubate at 4 °C for 30 min; then wash the cells twice with 0.5 mL of flow buffer (centrifuge at 400 xg at room temperature for 5 min), resuspend in 0.5 mL of PBS buffer, and perform flow cytometry detection. The obtained results are as Figure 5 shown.

[0094] The results show that the NK cells obtained in Example 2 express more inflammatory factors, and it can be speculated that they have better anti-tumor effects after being transfused into the body.

[0095] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of a combination of IL-12, IL-15 and IL-18 cytokines and IL-2 and / or IL-15 cytokines in the in vitro expansion culture of NK cells.

2. An in vitro expansion culture of NK cells, characterized in that: Including separate activation cultures and expansion cultures, The activated culture comprises a cell culture medium and a first active component added to the cell culture medium, wherein the first active component comprises IL-12, IL-15 and IL-18 cytokines; The expansion culture comprises a cell culture medium and a second active component added to the cell culture medium, wherein the second active component comprises IL-2 and / or IL-15 cytokines.

3. The NK cell in vitro expansion culture according to claim 2, characterized in that: In the activated culture, the concentration of the first active component is: IL-12 cytokine 10-50 ng / ml, IL-15 cytokine 1-100 ng / ml, IL-18 cytokine 50-100 ng / ml.

4. The NK cell in vitro expansion culture according to claim 2, characterized in that: In the expansion culture, the concentration of IL-2 cytokine is 100-1000 IU / ml, and the concentration of IL-15 cytokine is 1-50 ng / ml.

5. A method for in vitro expansion and cultivation of NK cells, characterized in that: The method comprises the following steps: using a combination of IL-12, IL-15 and IL-18 cytokines and IL-2 and / or IL-15 cytokines to culture NK cells in vitro.

6. The method for in vitro expansion and cultivation of NK cells according to claim 5, characterized in that: The following steps are involved: S1, collect peripheral blood and separate peripheral blood mononuclear cells and plasma; S2, heat-inactivating the plasma to obtain inactivated plasma; S3, resuspending the peripheral blood mononuclear cells and inoculating them in an activation medium and culturing them for 1-3 days; the activation medium comprises IL-12, IL-15 and IL-18 cytokines and 8-12 vol% inactivated plasma; S4, resuspending the cells obtained in step S3 in an expansion medium, and carrying out expansion culture for 4-6 days; the expansion medium comprises IL-2 and / or IL-15 cytokines; S5, continue culturing the cell suspension in step S4 for 4-7 days, and collect NK cells by centrifugation.

7. The method for in vitro expansion and cultivation of NK cells according to claim 6, characterized in that: In step S2, the heat inactivation treatment is: treating at 50-60°C for 20-40 minutes.

8. The method for in vitro expansion and cultivation of NK cells according to claim 6, characterized in that: In step S3, the cell seeding density is 2-4×10 6 cells / ml.

9. The method for in vitro expansion and cultivation of NK cells according to claim 6, characterized in that: In step S4, the cell density is maintained at 1-1.2×10 6 cells / ml.

10. Use of NK cells obtained by the in vitro expansion and culture method of NK cells according to any one of claims 5 to 9 in the preparation of drugs for preventing and / or treating tumors.

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