NK cell culture method and application of obtained NK cells in removing senescent cells and tumor cells

Abstract

The application belongs to the technical field of NK cells, and discloses an NK cell culture method and application of obtained NK cells in removing senescent cells and tumor cells. The NK cell culture method comprises the following steps: S1, coating the bottom of a culture container with a coating solution; S2, resuspending peripheral blood mononuclear cells and then adding them into the coated culture container obtained in S1 for culture; the coating solution in S1 contains CD16 antibody solution and CD244 antibody solution; and the components of the first activation culture medium contain IFN-gamma and OK432. The coating solution in S1 contains CD16 antibody solution and CD244 antibody solution, and the obtained NK cells have high survival rate and high activity; OK432 in the components of the first activation culture medium in S2 can simulate bacterial infection signals, IFN-gamma can activate NK cells through different signal pathways, and OK432 can enhance the cell activity of NK cells; the obtained NK cells have high activity, long life span and high response capacity, and therefore have high efficiency in removing senescent cells and tumor cells.

Description

Technical Field

[0001] This invention relates to the field of NK cell technology, and more specifically, to NK cell culture methods and the application of the resulting NK cells in clearing senescent cells and tumor cells. Background Technology

[0002] Natural killer (NK) cells are the core effector cells of the human innate immune system. Without antigen presentation, they can recognize and kill abnormal target cells such as tumor cells and virus-infected cells by balancing activating and inhibitory receptors on their surface. They can also secrete cytokines such as interferon-γ and tumor necrosis factor-α to regulate the function of dendritic cells and T cells, playing a key role in anti-tumor, anti-infection and immune homeostasis maintenance.

[0003] Existing technology uses peripheral blood isolation and culture to culture NK cells, including the following steps: First, peripheral blood mononuclear cells (PBMCs) are isolated from peripheral venous blood using density gradient centrifugation (commonly using Ficoll separation medium); then, the bottom of the culture container is coated with coating medium to resuspend the PBMCs, which are then seeded into coated culture flasks. Serum-containing or serum-free culture medium and a combination of cytokines such as IL-2 and IL-15 are added, and the flasks are placed in an incubator for culture. During the culture process, fresh culture medium and cytokines are added every 2-3 days to maintain cell density; after 14-16 days of culture, an expanded NK cell population is obtained. This method uses IL-2 as the core stimulating factor, but long-term high concentrations of IL-2 can lead to downregulation of the IL-2 receptor (IL-2R) on the surface of NK cells, inhibiting the expression of NK cell surface activation receptors (such as NKG2D and DNAM-1), resulting in altered NK cell function, reduced activity, shorter lifespan, and lower responsiveness of the resulting NK cells, leading to low efficiency in clearing senescent cells. Summary of the Invention

[0004] This invention provides a method for culturing NK cells, resulting in NK cells with high activity and superior efficiency in clearing senescent and tumor cells compared to existing technologies.

[0005] Another object of the present invention is to provide the application of NK cells cultured by the NK cell culture method in clearing senescent cells and tumor cells.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: NK cell culture method includes the following steps: S1. Coat the bottom of the culture container with a coating solution; S2. Peripheral blood mononuclear cells are resuspended and then added to the culture container coated in S1 for culture. The coating solution in S1 contains CD16 antibody solution and CD244 antibody solution; In S2, peripheral blood mononuclear cells are resuspended using a first activation medium containing IFN-γ and OK432.

[0007] The NK cell culture method described in this invention has the following technical effects: The coating solution in S1 contains CD16 antibody solution and CD244 antibody solution. CD16 receptor initially activates NK cells, while CD244 exerts a synergistic effect with the CD16 signaling pathway by regulating the intracellular adaptor protein SAP / EAT-2, thereby providing a strong co-stimulatory signal, stimulating NK cells to fully activate and initiate the proliferation program, promoting the activation and proliferation of NK cells, resulting in NK cells with high viability and strong activity.

[0008] In the first activation medium component of S2: (a) OK432 can mimic bacterial infection signals, activating members of the Toll-like receptor family (mainly TLR4) to trigger downstream signaling pathways such as NF-κB and MAPK. Furthermore, IFN-γ can bind to the highly expressed receptor IFN-γR on the surface of NK cells, thereby initiating the downstream JAK-STAT1 signaling pathway. NK cells can be activated through different signaling pathways. (b) OK432 can also upregulate the expression of NK cell surface activation receptors (such as NKG2D and DNAM-1), promoting NK cells to enter an activated state and enhancing their cellular activity. The resulting NK cells exhibit high activity, long lifespan, and high responsiveness, thus demonstrating high efficiency in clearing senescent and tumor cells.

[0009] Preferably, the cultivation in S2 specifically involves transferring the cultivation container to an incubator and sequentially adding the second activation medium and the amplification medium for cultivation; the components of the second activation medium in S2 contain IL-2, IL-12, IL-15, IL-18 and IL-21.

[0010] The NK cells obtained by this invention have high efficiency in clearing senescent cells and tumor cells. In the second activation medium in S2, on the one hand, the combination of IL-12, IL-15, and IL-18 cytokines can stimulate NK cells, transforming them into cytokine-induced memory-like NK cells. Memory-like NK cells have high activity, longer lifespan, and stronger secondary response capabilities, thus achieving high efficiency in clearing senescent cells. On the other hand, IL-21 in the second activation medium can promote the expression of NK cell granzyme and perforin, thus achieving high efficiency in clearing tumor cells.

[0011] More preferably, the NK cell culture method is characterized in that the concentrations of each component of the second activation medium in S2 are IL-2 100~2000 IU / mL, IL-12 0.1~200 ng / mL, IL-15 0.1~200 ng / mL, IL-18 0.1~200 ng / mL and IL-21 0.1~200 ng / mL.

[0012] Preferably, the concentration of CD16 antibody solution in the S1 coating solution is 0.1~5 μg / mL and the concentration of CD244 antibody solution is 0.1~5 μg / mL.

[0013] Preferably, the amplification medium in S2 contains IL-2 and IL-15.

[0014] More preferably, the concentrations of each component in the amplification medium described in S2 are: IL-2 100~2000 IU / mL, IL-15 1~200 ng / mL.

[0015] Preferably, the concentrations of each component in the first activating medium in S2 are: IFN-γ 0.1~2 μg / mL and OK432 0.1~2 μg / mL.

[0016] Preferably, the culture in S2 is specifically as follows: on the 3rd and 5th days of culture, the second activation medium is added; during the period from the 7th to the 16th day of culture, the amplification medium is added every 1 to 2 days.

[0017] More preferably, after resuspending the peripheral blood mononuclear cells in S2, the density of peripheral blood mononuclear cells in the first activation culture medium is 0.5 × 10⁻⁶. 6 ~2.5×10 6 The peripheral blood mononuclear cell density was 0.6 × 10⁶ cells / mL after the addition of amplification culture medium. 6 ~1.0×10 6 cells / mL.

[0018] The present invention also provides the application of NK cells cultured by any of the above-described NK cell culture methods in clearing senescent cells and tumor cells. Attached Figure Description

[0019] Figure 1 The purity of NK cells obtained from Example 1, Comparative Example 1, and Comparative Example 2 is shown.

[0020] Figure 2 The expression of NKG2D obtained from the culture of Example 1, Comparative Example 1 and Comparative Example 2.

[0021] Figure 3The expression of NKp46 obtained from the culture of Example 1, Comparative Example 1 and Comparative Example 2.

[0022] Figure 4 The effect of NK cells cultured in Example 1, Comparative Example 1, and Comparative Example 2 on the expression level of senescence marker (SA-β-Gal) in senescent T cells.

[0023] Figure 5 The ELISA method was used to detect the killing effect of NK cells obtained from Example 1, Comparative Example 1 or Comparative Example 2 on tumor cells HCT-116.

[0024] Figure 6 The ELISA method was used to detect the killing effect of NK cells obtained from Example 1, Comparative Example 1 or Comparative Example 2 on MCF-7 tumor cells.

[0025] Figure 7 The LDH method was used to detect the killing effect of NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 on tumor cells HCT-116.

[0026] Figure 8 The LDH method was used to detect the killing effect of NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 on MCF-7 tumor cells. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments.

[0028] Example 1 NK cell culture method includes the following steps: S1. The bottom of the culture container is coated with a coating solution; the coating solution contains CD16 antibody solution and CD244 antibody solution, wherein the concentration of CD16 antibody solution is 1 μg / mL and the concentration of CD244 antibody solution is 2 μg / mL; S2. Peripheral blood mononuclear cells are resuspended and then added to the coated culture container obtained in S1 for culture. Specifically, the culture container is transferred to an incubator, and a second activation medium and an amplification medium are added sequentially. The second activation medium is added on days 3 and 5 of culture, and the amplification medium is added every 1-2 days from days 7 to 16. After resuspending the peripheral blood mononuclear cells in S2, the peripheral blood mononuclear cell density in the first activation medium is 0.5 × 10⁻⁶ cells / day. 6 ~2.5×10 6 The number of cells / mL and the peripheral blood mononuclear cell density after amplification culture were both 0.6 × 10⁻⁶. 6 ~1.0×10 6 cells / mL; In S2, peripheral blood mononuclear cells are resuspended using a first activation medium containing IFN-γ and OK432, with IFN-γ concentration of 1 μg / mL and OK432 concentration of 1.5 μg / mL.

[0029] The second activation medium in S2 contains IL-2, IL-12, IL-15, IL-18 and IL-21, with IL-2 concentration of 800 IU / mL, IL-12 concentration of 100 ng / mL, IL-15 concentration of 100 ng / mL, IL-18 concentration of 50 ng / mL and IL-21 concentration of 50 ng / mL. The amplification medium contains IL-2 and IL-15, with IL-2 concentration of 600 IU / mL and IL-15 concentration of 150 ng / mL.

[0030] Example 2 This embodiment is the second embodiment of the present invention. The difference from embodiment 1 is that in the NK cell culture method, the concentration of CD16 antibody solution in the coating solution in S1 is 0.1 μg / mL and the concentration of CD244 antibody solution is 5 μg / mL.

[0031] Example 3 This embodiment is the third embodiment of the present invention. The difference from embodiment 1 is that in the NK cell culture method, the concentration of CD16 antibody solution in the coating solution in S1 is 5 μg / mL and the concentration of CD244 antibody solution is 0.1 μg / mL.

[0032] Example 4 This embodiment is the fourth embodiment of the present invention. Unlike embodiment 1, in the NK cell culture method, the concentration of IFN-γ in the first activation medium in S2 is 0.1 μg / mL and the concentration of OK432 is 2 μg / mL.

[0033] Example 5 This embodiment is the fifth embodiment of the present invention. Unlike embodiment 1, in the NK cell culture method, the concentration of IFN-γ in the first activation medium in S2 is 2 μg / mL and the concentration of OK432 is 0.1 μg / mL.

[0034] Example 6 This embodiment is the sixth embodiment of the present invention. The difference from embodiment 1 is that the concentration of IL-2 in the second activation medium in S2 is 2000 IU / mL, the concentration of IL-12 is 0.1 ng / mL, the concentration of IL-15 is 0.1 ng / mL, the concentration of IL-18 is 200 ng / mL, and the concentration of IL-21 is 0.1 ng / mL. The amplification medium in S2 has an IL-2 concentration of 100 IU / mL and an IL-15 concentration of 200 ng / mL.

[0035] Example 7 This embodiment is the 7th embodiment of the present invention. The difference from embodiment 1 is that the concentration of IL-2 in the second activation medium in S2 is 100 IU / mL, the concentration of IL-12 is 200 ng / mL, the concentration of IL-15 is 200 ng / mL, the concentration of IL-18 is 0.1 ng / mL, and the concentration of IL-21 is 200 ng / mL. The amplification medium in S2 has an IL-2 concentration of 2000 IU / mL and an IL-15 concentration of 1 ng / mL.

[0036] Comparative Example 1 This comparative example is the first comparative example of the present invention. Unlike Example 1, the S1 coating solution contains only CD244 antibody solution and does not contain CD16 antibody solution.

[0037] Comparative Example 2 This comparative example is the second comparative example of the present invention. Unlike Example 1, the S1 first activation culture medium contains only IFN-γ and does not contain OK432.

[0038] Comparative Example 3 This comparative example is the third comparative example of the present invention. Unlike Example 1, no second activation medium was added during the S2 culture process.

[0039] Comparative Example 4 This comparative example is the fourth comparative example of the present invention. Unlike Example 1, in the NK cell culture method, the concentration of CD16 antibody solution in the coating solution in S1 is 0.05 μg / mL and the concentration of CD244 antibody solution is 6 μg / mL.

[0040] Comparative Example 5 This comparative example is the fifth comparative example of the present invention. Unlike Example 1, in the NK cell culture method, the concentration of CD16 antibody solution in the coating solution in S1 is 6 μg / mL and the concentration of CD244 antibody solution is 0.05 μg / mL.

[0041] Comparative Example 6 This comparative example is the sixth comparative example of the present invention. Unlike Example 1, in the NK cell culture method, the concentration of IFN-γ in the first activation medium in S2 is 0.05 μg / mL and the concentration of OK432 is 3 μg / mL.

[0042] Comparative Example 7 This comparative example is the 7th comparative example of the present invention. Unlike Example 1, in the NK cell culture method, the concentration of IFN-γ in the first activation medium in S2 is 3 μg / mL and the concentration of OK432 is 0.05 μg / mL.

[0043] Performance testing: I. NK cell survival rate The viability of NK cells obtained from the various embodiments and comparative examples was detected using an NC-200 cell counter, as follows: Connect the NC-200 cell counter to the computer and turn on the power. Turn on the computer and double-click the cell counter icon to open the software. On the software interface, select the protocol matching the cell sample and enter the dilution factor. Take 50µL of NK cells obtained from any embodiment or comparative example on days 7 and 14 as the cell sample stock solution and add it to a 1.5mL EP tube. Add 50µL of DPBS (dilution factor 2), mix by pipetting 5 to 8 times, and count the cells. Insert the Via1-Cassette below the liquid surface at the bottom of the EP tube and press the white stopcock until it is flush with the transparent edge. Insert the Via1-Cassette containing the cell sample stock solution into the NC-200 cell counter to obtain the live cell count result, thereby obtaining the NK cell viability result.

[0044] The survival rates of NK cells obtained from Examples 1-7 and Comparative Examples 1-7 are shown in Table 1.

[0045] Table 1. Survival rates of NK cells obtained from Examples 1-7 and Comparative Examples 1-7

[0046] II. NK cell purity Take 5 × 10 5In Example 1, Comparative Example 1, or Comparative Example 2, NK cells cultured were centrifuged at 600 ×g at room temperature for 5 min, and the supernatant was discarded. The cells were then stained for 30 min with BV421-labeled anti-CD56 antibody, PE-labeled anti-CD3 antibody, APC-labeled anti-CD314 (NKG2D) antibody, and PE / Cyanine7-labeled CD335 (NKp46) antibody. After staining, the cells were centrifuged at 600 ×g at room temperature for 5 min, the supernatant was discarded, and the cells were washed three times with PBS. The cells were resuspended in 200 μL of MACS buffer and analyzed by flow cytometry. The population expressing CD3-PE negative and CD56-BV421 positive was identified as NK cells (i.e., CD3-CD56+), and the population expressing both CD3-PE and CD56-BV421 positive was identified as NKT cells (i.e., CD3+CD56+).

[0047] Figure 1 The purity (CD3-CD56+ %) of NK cells obtained from Example 1, Comparative Example 1, and Comparative Example 2 was determined from... Figure 1 It can be seen that the purity of NK cells obtained from Example 1 culture was 92.6%, which was much higher than that of Comparative Example 1 (88.1%) and Comparative Example 2 (80.0%).

[0048] III. NK Cell Activity 1. NK cells' response to NKG2D expression Take 5 × 10 5 NK cells obtained from Examples 1, 1, or 2 were centrifuged at 600 ×g at room temperature for 5 min, and the supernatant was discarded. The cells were then stained for 30 min with BV421-labeled anti-CD56 antibody, PE-labeled anti-CD3 antibody, APC-labeled anti-CD314 (NKG2D) antibody, and PE / Cyanine7-labeled CD335 (NKp46) antibody. After staining, the cells were centrifuged at 600 ×g at room temperature for 5 min, the supernatant was discarded, and the cells were washed three times with PBS. The cells were resuspended in 200 μL of MACS buffer and analyzed by flow cytometry. Histograms of NKG2D expression and MFI values ​​were obtained under the NK cell phylogenetic tree (i.e., CD3-CD56+).

[0049] Figure 2 The expression of NKG2D was obtained from the cultures of Example 1, Comparative Example 1, and Comparative Example 2. From... Figure 2As can be seen, the MFI value of NKG2D on the surface of the NK cells cultured in Example 1 was 158766, which was much higher than that of Comparative Example 1 and Comparative Example 2. NKG2D is an important activating receptor on the surface of NK cells. The recognition of senescent cells depends on the surface activating receptor of NK cells. The NK cells cultured in Example 1 have a high expression level of NKG2D, which can enhance the killing activity of NK cells and clear abnormal cells.

[0050] 2. NK cells' response to NKp46 expression Take 5 × 10 5 NK cells obtained from Examples 1, 1, or 2 were centrifuged at 600 ×g at room temperature for 5 min, and the supernatant was discarded. The cells were then stained for 30 min with BV421-labeled anti-CD56 antibody, PE-labeled anti-CD3 antibody, APC-labeled anti-CD314 (NKG2D) antibody, and PE / Cyanine7-labeled CD335 (NKp46) antibody. After staining, the cells were centrifuged at 600 ×g at room temperature for 5 min, the supernatant was discarded, and the cells were washed three times with PBS. The cells were resuspended in 200 μL of MACS buffer and analyzed by flow cytometry. Histograms of NKp46 expression and MFI values ​​were obtained under the NK cell phylogenetic tree (i.e., CD3-CD56+).

[0051] Figure 3 The expression of NKp46 was obtained from the cultures of Example 1, Comparative Example 1, and Comparative Example 2. From... Figure 3 Can As can be seen, the MFI value of NKp46 on the surface of the NK cells cultured in Example 1 was 97964, which was much higher than that in Comparative Examples 1 and 2, indicating that the NK cells cultured in Example 1 had a high expression level of NKp46. NKp46, as a member of the natural cytotoxic receptor (NCR), plays a crucial role in recognizing viral proteins (such as influenza virus, hepatitis C virus, etc.) and some tumor cells. Therefore, high expression of NKp46 on the surface of NK cells can enhance the killing activity of NK cells against viruses and tumor cells.

[0052] IV. The effect of NK cells in clearing senescent cells NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 were co-cultured with senescent T cells, and the expression levels of T cell senescence markers (SA-β-Gal) were detected, as detailed below: Senescent T cells were labeled with Cell Trace Violet, incubated in a CO2 incubator at 37°C in the dark for 10 minutes with shaking halfway through, then the staining was stopped by adding complete culture medium. After centrifugation at 600 × g for 5 min at room temperature, the cells were washed once and counted again. Cells were then treated with 1 × 10⁻⁶ culture medium. 6Labeled senescent T cells were resuspended at a density of cells / mL and seeded into 48-well plates at a volume of 100 μL per well. NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 were added at an effector-to-target ratio of 1:1 and co-cultured overnight.

[0053] Changes in T cell senescence markers were detected. After overnight culture, cells were centrifuged at 600 ×g at room temperature for 5 min, the supernatant was discarded, and cells were stained with APC-Cy7-labeled cell dye for 30 min. After centrifugation at 600 ×g at room temperature for 5 min, the supernatant was discarded, and cells were washed three times with PBS. Cells were fixed with 2% paraformaldehyde for 10 min, centrifuged at 600 ×g at room temperature for 5 min, the supernatant was discarded, and cells were washed three times with PBS. Cells were stained with X-Gal senescence assessment reagent at 37℃ for 2 h to detect changes in senescence markers. After centrifugation at 600 ×g at room temperature for 5 min, the supernatant was discarded, and cells were washed three times with MACS buffer. Cells were resuspended in 200 μL MACS buffer and flow cytometry analysis was performed to obtain the expression levels of the T cell senescence marker (SA-β-Gal).

[0054] Figure 4 The expression levels of the T cell senescence marker (SA-β-Gal) after co-culturing NK cells and T cells obtained from Examples 1, 1, and 2 were analyzed. Figure 4 It can be seen that after co-culturing NK cells and T cells obtained in Example 1, the expression level of T cell senescence marker (SA-β-Gal) was much lower than that in Comparative Example 1 and Comparative Example 2. The inhibitory effect of NK cells obtained in Example 1 on the expression of T cell senescence marker (SA-β-Gal) was much better than that in Comparative Example 1 and Comparative Example 2, and its effect on clearing senescent cells was much better than that in Comparative Example 1 and Comparative Example 2.

[0055] V. The effect of NK cells in eliminating tumor cells The NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 were co-cultured with tumor cells HCT-116 and MCF-7. The killing effect of NK cells on tumor cells was detected by ELISA (IFN-γ) and LDH methods.

[0056] 1. ELISA method to detect the killing effect of NK cells on tumor cells. This application judges the effect of NK cells in clearing tumor cells by detecting the IFN-γ content in the supernatant after NK cells and tumor cells are co-cultured. The principle is that when NK cells recognize and attack tumor cells, NK cells are activated and will secrete a large amount of IFN-γ. Therefore, the more IFN-γ in the supernatant, the stronger the clearing effect.

[0057] The specific testing methods are as follows: Use culture medium at 1×10 5 Tumor cells HCT-116 and MCF-7 were resuspended at a density of cells / mL and seeded into 96-well plates at a volume of 100 μL per well. NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 were added at an effector-to-target ratio of 5:1 and co-cultured overnight. After centrifugation at 600 × g at room temperature for 5 min, the supernatant was collected, and the IFN-γ content in the co-culture supernatant was detected using the BD OptEIA Human IFN-γ ELISA Set kit. The procedure included the following steps: (1) Reagent preparation: Coating solution is 0.1M sodium carbonate (pH 9.5); blocking solution is PBS + 10% FBS; elution solution is PBS + 0.05% Tween; chromogenic substrate is BD Pharmingen™ TMB Substrate Reagent Set.

[0058] (2) Coating Antibody: According to the kit requirements, the coating antibody (Capture Antibody Purified Anti) should be applied. Human IFN-γ was diluted 1:250 with coating solution, 100 μL / well, gently shaken and placed in a 4°C refrigerator overnight.

[0059] (3) Washing the plate: Discard the coating solution and wash the plate with elution solution, 200 μL / well, 3×1min (shake on a micro shaker each time you wash the plate).

[0060] (4) Blocking: Block with blocking solution, 200 μL / well, and let stand at room temperature for 1 hour.

[0061] (5) Washing the plate: Discard the blocking solution and wash the plate with the elution solution, 200 μL / well, 3×1min (shake the plate on a micro shaker each time you wash the plate).

[0062] (6) Dilution of standards and samples: In standard lyophilized vials (Recombinant Human IFN) Add 1 mL of deionized water to the γ-Lyophilized Standard, let stand for 15-20 min, vortex to mix to obtain the standard stock solution. Dilute with blocking buffer to obtain a standard solution with a theoretical concentration of 600 pg / mL. Then dilute by twofold to obtain seven other dilution grades (300 pg / mL, 150 pg / mL, 75 pg / mL, 37.5 pg / mL, 18.8 pg / mL, 9.4 pg / mL, and 4.7 pg / mL). Use the blocking buffer as the standard solution with a theoretical concentration of 0 pg / mL. Dilute the test sample with the blocking buffer.

[0063] (7) Adding samples: Start by adding the standard and the sample, 100 μL / well, mix well and let stand at room temperature for 2 hours.

[0064] (8) Washing the plate: Discard the sample solution and wash the plate with elution buffer, 200 μL / well, 5 × 1 min (shake on a micro shaker each time you wash the plate).

[0065] (9) Add antibody and SAv-HRP: Prepare the antibody mixture 15 minutes in advance (Detection Antibody Biotin Anti). Human IFN-γ and SAv-HRP were diluted 1:250, 100 μL / well, mixed well and incubated at room temperature for 1 hour.

[0066] (10) Washing: Discard the antibody and wash the plate with elution buffer, 200 μL / well, 7 × 1 min (shake on a micro-shaker after each wash).

[0067] (11) Color development and termination: The color development substrate should be prepared fresh and used immediately. The ratio of A to B is 1:1 (volume ratio), 100 μL / well. After adding the color development reagent, let it stand at room temperature in the dark. Start timing from the time the substrate is added. After 30 min, add 1M H2SO4 stop solution (50 μL / well) to terminate the color development.

[0068] (12) Read the plate and save the original results: Detect the absorbance at OD450 (570) using an ELISA reader. Build a standard curve based on the serially diluted standard wells. If the R² value of the standard curve is ≥ 0.99, then the standard curve fit meets the requirements.

[0069] The killing effect of NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 on HCT-116 tumor cells is shown in the figure. Figure 5 The killing effect of MCF-7 on tumor cells is shown in the figure. Figure 6 .from Figure 5 and Figure 6 It can be seen that the IFN-γ content in the co-culture supernatant of the NK cells obtained in Example 1 after co-culturing with tumor cells HCT-116 and MCF-7 is much higher than that in Comparative Example 1 and Comparative Example 2. Therefore, the killing effect of the NK cells obtained in Example 1 on tumor cells is much higher than that in Comparative Example 1 and Comparative Example 2.

[0070] 3. The LDH method was used to detect the killing effect of NK cells on tumor cells, as detailed below: Use culture medium at 1×10 5Tumor cells HCT-116 and MCF-7 were resuspended at a density of cells / mL and seeded into 96-well plates at a volume of 100 μL per well. NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 were added at an effector-to-target ratio of 8:1, 4:1, or 2:1 and co-cultured overnight. The next day, 10 μL of lysis buffer was added to each well, and cells were lysed for 1 hour. After centrifugation at 600 ×g at room temperature for 5 min, the supernatant was collected. 50 μL of the cell supernatant was added to 50 μL of substrate from the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, and the reaction was carried out for 30 min. The absorbance at OD490 was measured using a microplate reader, and the killing rate of NK cells against tumor cells HCT-116 and MCF-7 was calculated based on the absorbance. The formula for calculating the killing rate is as follows: lethality % = ×100% In the above formula, the target cell spontaneous release group refers to target cells + culture medium, the effector cell spontaneous release group refers to NK cells + culture medium, the target cell maximum release group refers to target cells + culture medium + lysis buffer, and the culture medium lysis buffer release group refers to culture medium + lysis buffer.

[0071] The killing effect of NK cells obtained from Example 1, Comparative Example 1, or Comparative Example 2 on HCT-116 tumor cells is shown in the figure. Figure 7 The killing effect of MCF-7 on tumor cells is shown in the figure. Figure 8 .from Figure 7 and Figure 8 It can be seen that, under the same effect-to-target ratio, the killing rate of NK cells obtained in Example 1 against tumor cells HCT-116 and MCF-7 is much higher than that of Comparative Example 1 and Comparative Example 2.

[0072] The above description is merely an embodiment of the present invention and does not limit the scope of patent protection. Any non-substantial changes or substitutions made by those skilled in the art based on the present invention will still fall within the scope of patent protection.

Claims

1. NK cell culture method, including the following steps: S1. Coat the bottom of the culture container with a coating solution; S2. Peripheral blood mononuclear cells are resuspended and then added to the culture container coated in S1 for culture. Its features are, The coating solution in S1 contains CD16 antibody solution and CD244 antibody solution; In S2, peripheral blood mononuclear cells are resuspended using a first activation medium containing IFN-γ and OK432.

2. The NK cell culture method according to claim 1, characterized in that, The cultivation in S2 specifically involves transferring the cultivation container to an incubator and sequentially adding the second activation medium and the amplification medium for cultivation; the components of the second activation medium in S2 include IL-2, IL-12, IL-15, IL-18 and IL-21.

3. The NK cell culture method according to claim 1, characterized in that, The concentration of CD16 antibody solution in the S1 coating solution is 0.1~5 μg / mL, and the concentration of CD244 antibody solution is 0.1~5 μg / mL.

4. The NK cell culture method according to claim 1, characterized in that, The amplification medium described in S2 contains IL-2 and IL-15.

5. The NK cell culture method according to claim 3, characterized in that, The concentrations of each component in the amplification medium described in S2 are: IL-2 100~2000 IU / mL, IL-1 5 1~200 ng / mL.

6. The NK cell culture method according to claim 1, characterized in that, The concentrations of each component in the first activation medium in S2 are: IFN-γ 0.1~2 μg / mL and OK432 0.1~2 μg / mL.

7. The NK cell culture method according to claim 2, characterized in that, The concentrations of each component in the second activation medium in S2 are IL-2 100~2000 IU / mL, IL-12 0.1~200 ng / mL, IL-15 0.1~200 ng / mL, IL-18 0.1~200 ng / mL and IL-21 0.1~200 ng / mL.

8. The NK cell culture method according to claim 1, characterized in that, The S2 culture process involves adding the second activation medium on days 3 and 5 of the culture, and adding the amplification medium every 1 to 2 days from day 7 to day 16 of the culture.

9. The NK cell culture method according to claim 8, characterized in that, After resuspending the peripheral blood mononuclear cells in S2, the density of peripheral blood mononuclear cells in the first activation culture medium was 0.5 × 10⁻⁶. 6 ~2.5×10 6 The peripheral blood mononuclear cell density was 0.6 × 10⁶ cells / mL after the addition of amplification culture medium. 6 ~1.0×10 6 cells / mL.

10. The use of NK cells cultured by the NK cell culture method according to any one of claims 1 to 9 in clearing senescent cells and tumor cells.

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