Method for improving killing activity of NK cells

Abstract

The invention discloses a method for improving killing activity of NK cells, and belongs to the technical field of cellular immunity. Carrying out segmented in-vitro culture on the separated NK cells by adopting a specific basic induction culture medium, an activation induction culture medium and an amplification culture medium, wherein recombinant human insulin and sodium selenite are added into the basic induction culture medium; ganoderma lucidum polysaccharides, eicosapentaenoic acid, IL-15 and IL-18 are added into the activation and induction culture medium. Salidroside, tea polyphenol, low molecular weight chitosan, IL-2 (interleukin-2), IL-21 (interleukin-21) and SCF (mesenchymal stem cells) are added into the amplification culture medium. According to the method, the killing ability of the NK cells obtained through in-vitro culture on tumor cells and the in-vitro proliferation effect of the NK cells are remarkably improved, the problems that in the prior art, the in-vitro proliferation efficiency of the NK cells is low, and cell activity is difficult to consider are solved, and a foundation is laid for clinical transformation of NK cell immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of cellular immunity technology, in particular to a method for improving the killing activity of NK cells. Background Art

[0002] Malignant tumors pose a serious threat to human health. Currently, clinical treatments primarily include surgery, radiotherapy, chemotherapy, and immunotherapy. Among these, tumor immunotherapy, which activates or enhances the body's immune system to identify and kill tumor cells, has become a significant breakthrough following traditional therapies. In recent years, immunotherapy, represented by immune checkpoint inhibitors and CAR-T cell therapy, has made significant progress. However, it still faces challenges in the treatment of solid tumors, such as limited response rates and significant toxic side effects. Therefore, the development of new, safer and more effective immunotherapy strategies is of great clinical value.

[0003] Natural killer (NK) cells, as core effector cells of the innate immune system, play a key role in anti-tumor immune surveillance. Unlike T cells, NK cells can rapidly recognize and kill tumor cells through "self-loss" and "self-induction" mechanisms without prior antigen sensitization. They also secrete effector molecules such as interferon-γ (IFN-γ) and granzyme B to modulate the immune microenvironment. This unique biological property gives NK cells significant advantages in tumor immunotherapy: ① They are not restricted by the major histocompatibility complex (MHC); ② They can avoid serious side effects such as cytokine storms; and ③ They show therapeutic potential for both hematologic and solid tumors.

[0004] However, the clinical application of NK cells still faces key bottlenecks: First, the proportion of NK cells in peripheral blood only accounts for 5-10% of lymphocytes, making it difficult to directly obtain the required therapeutic dose; second, existing in vitro expansion technologies generally have problems such as low cell proliferation efficiency and difficulty in maintaining killing activity. Although studies have attempted to expand NK cells through cytokine combinations (such as IL-2 / IL-15) or feeder cell co-culture, these methods may lead to functional exhaustion or introduce safety risks due to the introduction of exogenous cells, which greatly limits the quality and stability of cell products.

[0005] Therefore, developing a new culture system that can simultaneously achieve efficient expansion and functional enhancement of NK cells, breaking through the dilemma of balancing proliferation efficiency and cell activity in existing technologies, is of great significance for promoting the clinical transformation of NK cell immunotherapy. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for increasing the cytotoxic activity of NK cells to address the problems of the prior art. The present invention significantly enhances the tumor cell-killing ability of NK cells cultured in vitro and the in vitro proliferation of NK cells, resolving the prior art issues of low in vitro expansion efficiency and difficulty balancing NK cell activity, thus laying the foundation for the clinical application of NK cell immunotherapy.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a culture medium for improving the killing activity of NK cells, comprising a basic induction culture medium and an activation induction culture medium;

[0009] The basic induction medium components are 1-2 μg / mL recombinant human insulin + 0.01-0.03 μg / mL sodium selenite + X-VIVO 15 medium;

[0010] The components of the activation induction culture medium are 10-15 μg / mL Ganoderma lucidum polysaccharide+5-8 μg / mL eicosapentaenoic acid+60-80 ng / mL IL-15+120-150 ng / mL IL-18+X-VIVO 15 culture medium.

[0011] Optionally, expansion medium is also included.

[0012] Optionally, the components of the expansion culture medium are 10-20 μg / mL salidroside + 2-8 μg / mL tea polyphenols + 2-6 μg / mL low molecular weight chitosan + 400-600 IU / mL IL-2 + 80-120 IU / mL IL-21 + 20-30 ng / mL SCF + RPMI 1640 culture medium.

[0013] The present invention also provides application of the culture medium in improving the killing activity of NK cells.

[0014] The present invention also provides an in vitro culture method for improving the killing activity of NK cells, comprising the steps of using the culture medium to culture NK cells in segmented in vitro.

[0015] Optionally, the method includes the following steps:

[0016] Performing basic induction culture on NK cells using the basic induction culture medium;

[0017] After the basic induction culture is completed, the basic induction medium is replaced with the activation induction medium for activation culture.

[0018] Optionally, after the activation culture is completed, the activation induction medium is replaced with an expansion medium for expansion culture.

[0019] Optionally, the basic induction culture time is 2 days, and the activation culture time is 7 days.

[0020] Optionally, the expansion culture time is 3 days, and the expansion culture medium is replenished once on the second day of the expansion culture.

[0021] Optionally, the activation induction medium is replenished every 2-3 days during the activation culture process.

[0022] The present invention discloses the following technical effects:

[0023] The present invention significantly enhances the ability of NK cells cultured in vitro to kill tumor cells by designing specific culture medium components for three stages: basic induction, activation induction, and expansion culture. Furthermore, the synergistic effect of salidroside and tea polyphenols in the expansion culture medium significantly enhances the in vitro proliferation of NK cells, increasing the expansion multiple of NK cells in vitro to 436-452 times. This achieves efficient in vitro culture of NK cells and solves the problem of low in vitro expansion efficiency of NK cells in the prior art.

[0024] The present invention combines staged culture with regular culture medium replenishment (e.g., feeding every 2-3 days during the activation phase and on the second day during the expansion phase) to avoid cell function exhaustion while maintaining high cell activity. Insulin and sodium selenite during the basal induction phase (2 days) lay the metabolic foundation for subsequent activation, while IL-2 / IL-21 during the expansion phase further consolidates proliferation and function, forming a complete "induction-activation-amplification" chain.

[0025] All additives of the present invention (such as Ganoderma lucidum polysaccharide and low molecular weight chitosan) are natural or recombinant ingredients, do not require feeder cells, avoid the risk of exogenous contamination, meet clinical transformation requirements, and lay the foundation for the clinical transformation of NK cell immunotherapy. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The present invention provides a method for improving the killing activity of NK cells, which uses a specific basic induction medium, an activation induction medium and an expansion medium to culture the separated NK cells in vitro in sections.

[0032] The basic induction medium is supplemented with recombinant human insulin and sodium selenite; the activation induction medium is supplemented with ganoderma lucidum polysaccharide, eicosapentaenoic acid, IL-15 and IL-18; and the expansion medium is supplemented with salidroside, tea polyphenols, low molecular weight chitosan, IL-2, IL-21 and SCF.

[0033] In some specific embodiments of the present invention, the components of the basal induction medium include 1-2 μg / mL recombinant human insulin + 0.01-0.03 μg / mL sodium selenite + X-VIVO 15 medium; the components of the activation induction medium include 10-15 μg / mL Ganoderma lucidum polysaccharide + 5-8 μg / mL eicosapentaenoic acid + 60-80 ng / mL IL-15 + 120-150 ng / mL IL-18 + X-VIVO 15 medium; the components of the expansion medium include 10-20 μg / mL salidroside + 2-8 μg / mL tea polyphenols + 2-6 μg / mL low molecular weight chitosan + 400-600 IU / mL IL-2 + 80-120 IU / mL IL-21 + 20-30 ng / mL SCF + RPMI1640 medium.

[0034] The segmented in vitro culture includes first performing basic induction culture using a basic induction medium, then performing activation culture on NK cells using an activation induction medium to enhance the killing activity of NK cells, and finally performing expansion culture on the activated NK cells using an expansion medium to allow the NK cells to proliferate in large quantities.

[0035] Optionally, corresponding culture medium needs to be supplemented during the activation culture and the expansion culture.

[0036] Optionally, the corresponding culture medium is replenished every 2-3 days during the activation culture process; and the corresponding culture medium is replenished once during the expansion culture process.

[0037] In some specific embodiments of the present invention, the basic induction culture time is 2 days; the activation culture time is 7 days; and the expansion culture time is 3 days.

[0038] The NK cells obtained by the method of the present invention have significantly enhanced killing activity against tumor cells.

[0039] Optionally, the tumor cells include liver cancer cells and lung cancer cells.

[0040] The method of the present invention can significantly increase the proliferation rate of NK cells.

[0041] Reagents used in the examples of the present invention: recombinant human insulin was purchased from McLean, Ganoderma lucidum polysaccharide was purchased from Beijing Solaibao Technology Co., Ltd., low molecular weight chitosan, salidroside (≥98%, HPLC), and tea polyphenols (TP 98%) were purchased from Aladdin.

[0042] 1. Acquisition of NK cells

[0043] 20 mL of peripheral blood (approved by the ethics committee and with informed consent) was collected from subjects without autoimmune diseases, tumors, viral and bacterial infections, and heparin was added for anticoagulation. Peripheral blood was diluted with PBS buffer at a ratio of 1:1, and an equal volume of Ficoll separation solution was added to a sterile centrifuge tube in advance. The diluted peripheral blood was then added to the Ficoll separation solution (be careful not to shake or blow), and centrifuged at 500 × g in a centrifuge for 15 minutes. After carefully removing the upper plasma, the middle buffy coat cells were aspirated, and then washed twice with 3 times PBS buffer, and centrifuged again at 500 × g for 15 minutes. The precipitate was the peripheral blood mononuclear cells (PBMC). The PBMC cells were resuspended in X-VIVO 15 culture medium and precipitated using EasySep TM NK cells were isolated by human NK cell immunomagnetic bead sorting.

[0044] 2. NK cell culture in vitro

[0045] Table 1 shows the components of the basal induction medium, activation induction medium and expansion medium corresponding to Examples 1-3.

[0046] Table 1 Components of basal induction medium, activation induction medium and expansion medium corresponding to Examples 1-3

[0047]

[0048]

[0049] The isolated NK cells were prepared into 1×10 6 cells / mL of cell suspension; 10 mL of cell suspension was inoculated into a culture dish and placed in an incubator at 37°C and 5% CO2;

[0050] After culturing for 2 days, the activation induction medium of the corresponding example was replaced and the cell density was adjusted to 1×10 6 cells / mL, and continue to culture in a 37°C, 5% CO2 incubator;

[0051] On the 5th and 7th days, the corresponding activation induction medium was added and the cell density was adjusted to 1×10 6 cells / mL, and continue to culture in a 37°C, 5% CO2 incubator;

[0052] On day 9, the culture medium was replaced with the expansion medium of the corresponding example and the cell density was adjusted to 1×10 6 cells / mL, and continue to culture in a 37°C, 5% CO2 incubator;

[0053] On day 11, the corresponding expansion medium was added and the cell density was adjusted to 1×10 6 cells / mL, and continue to culture in a 37°C, 5% CO2 incubator;

[0054] On day 12, the culture was terminated and NK cells were collected for subsequent experiments.

[0055] Comparative Example 1-8 NK cell culture in vitro

[0056] The specific operation is the same as "2. In vitro culture of NK cells", except that the basal induction medium, activation induction medium, and expansion medium of Comparative Examples 1-8 are used respectively. The components of the basal induction medium, activation induction medium, and expansion medium of Comparative Examples 1-8 are shown in Table 2.

[0057] Table 2 Components of basal induction medium, activation induction medium and expansion medium corresponding to comparative examples 1-8

[0058]

[0059]

[0060] Verification Example 1: Expansion times of NK cells cultured with different culture methods

[0061] 1. Prepare the NK cells obtained in Examples 1-3 and Comparative Examples 1-8 into a cell suspension, and take 10 μL of the cell suspension and place it in a sterile centrifuge tube;

[0062] 2. Add 10 μL of 0.4% trypan blue staining solution to the centrifuge tube and mix the staining solution and cell suspension in a 1:1 ratio;

[0063] 3. After gently mixing, incubate at room temperature for 2 minutes, take 10 μL of the stained cell suspension and carefully add it dropwise to the counting chamber of the hemocytometer, ensuring that the droplets are evenly distributed throughout the counting chamber;

[0064] 4. Count the cells under a microscope and calculate the total number of cells. The results are shown in Table 3.

[0065] Table 3 Expansion times of NK cells by different culture methods

[0066]

[0067]

[0068] From the results in Table 3, it can be seen that the NK cell expansion effects of Examples 1-3 are significantly better than those of Comparative Examples 1-8, among which Example 3 has the most significant effect, with an expansion factor of up to 452. From the data of Comparative Examples 1-3, it can be seen that salidroside and tea polyphenols have the greatest effect on the proliferation of NK cells, and changing the type and dosage of these two substances in the expansion medium will have a huge impact on the proliferation effect of NK cells.

[0069] By comparing the expansion effects of the examples and the comparative examples, it can be clearly seen that the culture medium provided in Examples 1-3 of the present invention has a significant synergistic promoting effect on the expansion of NK cells.

[0070] Verification Example 2: Cytotoxicity of NK cells cultured by different methods against cancer cells

[0071] 1. Tumor Cell Preparation

[0072] (1) HepG2 liver cancer cells and A549 lung cancer cells were cultured in culture flasks until the logarithmic growth phase, ensuring that the cells grew well and were not over-confluent;

[0073] (2) Digest the cells with trypsin (0.25% trypsin-EDTA). Once the cells are observed to be rounded, add complete culture medium to neutralize the trypsin.

[0074] (3) Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant;

[0075] (4) Resuspend the cells in an appropriate amount of culture medium, count them using a cell counting plate, and adjust the cell concentration to 1×10 5 cells / mL;

[0076] (5) The cell suspension was dispensed into 96-well plates, and 100 μL of cell suspension was added to each well (i.e., 1×10 4 cell);

[0077] (6) Place the 96-well plate in a 37°C, 5% CO2 incubator and culture for 24 hours or until the cells are completely attached.

[0078] 2. NK Cell Preparation

[0079] The NK cells cultured according to Examples 1-3 and Comparative Examples 1-8 were prepared into 1×10 6 cells / mL cell suspension.

[0080] 3. Cell Co-culture

[0081] (1) After the tumor cells adhered to the wall, carefully aspirate the culture medium and add 100 μL of the above-mentioned NK cell suspension;

[0082] (2) A blank control group was set up, with only 100 μL of tumor cell suspension added to each well without NK cells;

[0083] (3) Negative control group: only 100 μL of culture medium was added to each well without cells. Three replicate wells were set up for each treatment group to ensure the reliability of the experimental data.

[0084] 4. Incubation

[0085] The 96-well plate was placed in a 37°C, 5% CO2 incubator and incubated for 4 hours.

[0086] 5.CCK-8 assay

[0087] (1) After the incubation is completed, remove the original culture medium and add the culture medium containing CCK-8 reagent. Mix carefully to avoid bubbles.

[0088] (2) The 96-well plate was placed in the incubator and incubated for 3 hours. The optical density (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader to calculate the killing rate:

[0089] Killing rate (%) = OD value of blank control group - OD value of experimental group / OD value of blank control group × 100%.

[0090] The results are shown in Table 4.

[0091] Table 4 Cytotoxicity of NK cells cultured by different methods against cancer cells

[0092]

[0093] As shown in Table 4, the NK cells cultured in Examples 1-3 of the present invention showed significantly better cytotoxicity against liver cancer cells and lung cancer cells than those in Comparative Examples 1-8. The most significant effect was achieved in Example 2, with a 75.63% cytotoxicity against liver cancer cells and a 72.54% cytotoxicity against lung cancer cells. The data from Comparative Examples 1, 4-5, and 8 indicate that Ganoderma lucidum polysaccharides and eicosapentaenoic acid have the greatest impact on the cytotoxicity of NK cells. Changing the type and dosage of these two substances in the expansion medium significantly influences the cytotoxicity of NK cells, demonstrating that the two substances synergistically enhance the cytotoxicity of NK cells.

[0094] By comparing the amplification effects of the examples and the comparative examples, it can be clearly seen that the use of the culture medium provided in Examples 1-3 of the present invention for culturing has a significant synergistic promoting effect on improving the killing activity of NK cells.

[0095] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A culture medium for improving the killing activity of NK cells, characterized in that Including basic induction medium and activated induction medium; The basic induction medium components are 1-2 μg / mL recombinant human insulin + 0.01-0.03 μg / mL sodium selenite + X-VIVO 15 medium; The components of the activation induction culture medium are 10-15 μg / mL Ganoderma lucidum polysaccharide+5-8 μg / mL eicosapentaenoic acid+60-80 ng / mL IL-15+120-150 ng / mL IL-18+X-VIVO 15 culture medium.

2. The culture medium according to claim 1, characterized in that Expansion medium is also included.

3. The culture medium according to claim 2, characterized in that The components of the expansion culture medium are 10-20 μg / mL salidroside + 2-8 μg / mL tea polyphenols + 2-6 μg / mL low molecular weight chitosan + 400-600 IU / mL IL-2 + 80-120 IU / mL IL-21 + 20-30 ng / mL SCF + RPMI 1640 culture medium.

4. Use of the culture medium according to any one of claims 1 to 3 in improving the killing activity of NK cells.

5. An in vitro culture method for improving the killing activity of NK cells, characterized in that: The method comprises the steps of performing segmented in vitro culture of NK cells using the culture medium according to any one of claims 1 to 3.

6. The in vitro culture method according to claim 5, characterized in that The specific steps include: Performing basic induction culture on NK cells using the basic induction culture medium; After the basic induction culture is completed, the basic induction medium is replaced with the activation induction medium for activation culture.

7. The in vitro culture method according to claim 6, characterized in that After the activation culture is completed, the activation induction medium is replaced with an expansion medium for expansion culture.

8. The in vitro culture method according to claim 6, characterized in that The time for the basic induction culture is 2 days, and the time for the activation culture is 7 days.

9. The in vitro culture method according to claim 7, characterized in that The expansion culture lasts for 3 days, and the expansion culture medium is replenished once on the second day of the expansion culture.

10. The in vitro culture method according to claim 6, characterized in that: During the activation culture process, the activation induction medium is replenished every 2-3 days.

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