A method for culturing NK cells and its application

Abstract

This invention relates to the field of cell culture technology, and discloses a method for culturing NK cells and its applications. When NK cells enter a hypoxic environment, not only do their cytotoxic activity and proliferative capacity rapidly decline, but they also experience metabolic disorders such as impaired mitochondrial function, insufficient ATP production, and abnormal accumulation of reactive oxygen species (ROS), leading to functional failure and limiting their application in tumor therapy. Existing technologies mainly rely on constant or single-stage hypoxia induction, failing to improve the insufficient hypoxia adaptation of NK cells at the metabolic level. Therefore, this invention provides a method that uses repeated acclimatization culture with gradually decreasing oxygen concentration to allow NK cells to gradually adapt to the metabolic demands of a hypoxic environment. NK cells cultured using this method exhibit a more stable metabolic state and superior cytotoxic activity in a hypoxic environment, significantly enhancing their anti-tumor efficacy and solving the problem of metabolic imbalance and rapid functional loss of NK cells in the hypoxic tumor microenvironment in existing technologies.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically, to a method and application for culturing NK cells. Background Technology

[0002] Natural killer cells (NK cells) are an important component of the body's innate immune system. They have the ability to directly recognize and kill tumor cells and virus-infected cells without antigen pretreatment, and have broad application prospects in anti-tumor immunotherapy. NK92 cells, as an NK cell line with a well-established source, easy expansion and manipulation, have been widely used in cell immunotherapy research.

[0003] However, hypoxia is a typical characteristic of the tumor microenvironment. In tumor tissue, due to abnormal angiogenesis and metabolic disorders, local oxygen concentration is significantly reduced, often falling below 1%-3%. Numerous studies have shown that NK cells experience a rapid decline in cytotoxic activity upon entering a hypoxic tumor microenvironment, leading to severely limited killing function. Further research has revealed that hypoxia not only directly inhibits the effector function of NK cells but also induces mitochondrial dysfunction, decreased oxidative phosphorylation efficiency, and reduced metabolic flexibility by remodeling their energy metabolism, thereby limiting the sustained effector capacity of NK cells in the tumor microenvironment. This hypoxia-induced metabolic disorder is considered one of the important intrinsic mechanisms of NK cell functional decline, greatly limiting the application of NK cells in tumor therapy, drug screening, and clinical translation. Furthermore, existing research also indicates that the hypoxic environment plays a crucial role in regulating natural killer (NK) cell function. Therefore, it is necessary to investigate how to maintain or even enhance the metabolic function of NK cells under hypoxic conditions.

[0004] Currently, existing technologies disclose a method for inducing NK cells to acquire hypoxia adaptation in vitro. This method involves culturing NK cells under normoxic conditions followed by transfer to hypoxic conditions for further culturing, thereby obtaining NK cells with a certain anti-tumor ability. Existing technologies also disclose a method for culturing NK cells under continuous hypoxia (5% O2) conditions, which can achieve efficient expansion of NK cells and strong cytotoxic activity. However, existing technologies still have the following shortcomings: First, most methods use constant low-oxygen culture, which fails to simulate the dynamic oxygen concentration changes experienced by NK cells in the tumor microenvironment. Second, the lack of assessment of cell function during the reoxygenation phase after hypoxia adaptation leads to unstable induced cell function. Third, the NK cell killing activity induced by existing technologies is limited; Fourth, some methods have long culture cycles, are complex to operate, and have poor reproducibility, which are not conducive to their application in scientific research or clinical settings.

[0005] Fifth, existing technologies mainly focus on improving NK cell phenotype and killing function, lacking systematic regulatory strategies for the metabolic state of NK cells under hypoxic conditions. There is no method that can simultaneously maintain or enhance the energy metabolism level of NK cells in a hypoxic environment, thereby providing metabolic support for their continued anti-tumor function.

[0006] Therefore, it is still necessary to further study how to maintain the function and enhance the activity of NK cells under hypoxic conditions. Summary of the Invention

[0007] One of the objectives of this invention is to provide a cell culture method that enables NK cells to maintain strong cytotoxic activity and energy metabolism levels even under low oxygen concentrations.

[0008] This invention provides a method for culturing NK cells, which involves subjecting NK cells to multiple acclimatization cultures, wherein the oxygen concentration decreases in a gradient during each acclimatization culture, and the number of acclimatization cultures is greater than 1.

[0009] This invention reveals that hypoxia is a typical characteristic of the tumor microenvironment in solid tumors. NK cells, upon entering the tumor microenvironment, rapidly lose their killing ability due to hypoxia exposure, greatly limiting the application of immune cells, including NK cells, in tumor treatment. Currently, the anti-tumor effect of NK cells under hypoxic conditions still needs improvement. Based on this, this invention conducted extensive research and ultimately found that compared to known methods of single-stage acclimation from normoxic to a fixed hypoxic concentration (e.g., 1%), gradually acclimating NK cells using a gradient decrease in oxygen concentration multiple times allows the obtained NK cells to maintain significant cytotoxicity and energy metabolism levels even under hypoxic conditions (oxygen concentration ≤ 1%), particularly significantly enhancing their killing ability against non-small cell lung cancer target cells.

[0010] While existing techniques for culturing mesenchymal stem cells (MSCs) also teach the use of gradually decreasing oxygen concentrations, their aim is to maintain the stemness of MSCs in vitro or improve the quality and stability of their exosome secretion—an optimization strategy tailored to the biological characteristics of stem cells. This differs from the technical objective and challenges of this invention, which aims to enable NK cells to acquire functional adaptation to persistent, pathological hypoxia in the tumor microenvironment and to maintain or restore their cytotoxic function under hypoxic conditions.

[0011] Furthermore, MSCs and NK cells differ fundamentally in their response mechanisms to hypoxia. MSCs naturally originate from the hypoxic bone marrow environment, and hypoxia helps maintain their stemness and functional stability; while NK cells, as effector immune cells highly dependent on metabolic homeostasis, are generally believed in accordance with existing research to have their activation receptor expression, cytotoxic granule function, and energy metabolism inhibited by hypoxia, thereby weakening their antitumor activity. Therefore, the existing technology offers very limited guidance for obtaining the solution of this invention.

[0012] This invention first reoxygenates (restores to normoxic conditions) and then subjectes NK cells to hypoxia stimulation (rapidly placing them in a hypoxic environment) when testing their performance. This better simulates the acute hypoxia inhibition faced by NK cells as they enter the tumor microenvironment from the peripheral circulation, thus obtaining test results that are more consistent with actual application effects.

[0013] In the method of the present invention, the domestication and cultivation are carried out 3-5 times, and the domestication and cultivation time is 2-3 days each time.

[0014] In the method of this invention, the oxygen concentration gradient is reduced by 2-3% during each domestication and cultivation.

[0015] In the method of the present invention, when the number of domestication and cultivation is 3, the oxygen concentration during the first domestication and cultivation is 5%-6%.

[0016] Those skilled in the art can adjust the specific number of culture cycles and oxygen gradient settings based on the actual cell culture results, according to the culture conditions defined in this invention.

[0017] Preferably, in the method of the present invention, the number of acclimatization cultures is 3, the oxygen concentration during the first acclimatization culture is 5%, the oxygen concentration gradient during each acclimatization culture is reduced by 2%, and the duration of each acclimatization culture is 2 days, so that NK cells first begin to adapt to low oxygen, and then further tolerate low oxygen, and finally obtain cells in a low oxygen tolerant state.

[0018] In the method of the present invention, the NK cells are cultured to the logarithmic growth phase under an oxygen concentration of 20%-22% before being domesticated and cultured.

[0019] In the method of this invention, the NK cells are NK92 cells.

[0020] In this invention, NK cells can be cultured using NK cell culture media known in the art (e.g., α-MEM medium containing IL-2) and conventional culture temperatures (e.g., 37°C).

[0021] In this invention, the source of NK cells is not limited; they can be purchased or prepared using methods known in the art.

[0022] The present invention also provides an NK cell, which is obtained by the above-described method for culturing NK cells.

[0023] The NK cells of this invention exhibit higher physiological activity under hypoxic conditions, which can significantly enhance the tumor-killing effect.

[0024] The present invention also provides the application of the above-mentioned NK cells in the preparation of antitumor products.

[0025] The present invention also provides an anti-tumor product comprising the above-mentioned NK cells, preferably wherein the tumor is non-small cell lung cancer.

[0026] Preferably, the product of the present invention is a pharmaceutical product, such as an immune cell preparation.

[0027] The pharmaceutical products of the present invention may also include pharmaceutically acceptable excipients. The formulation is not limited, but is preferably an injection.

[0028] The medicine of the present invention can be prepared using conventional methods and may contain a therapeutically effective amount of a pharmacologically active ingredient or may also contain one or more pharmaceutically acceptable carriers.

[0029] The unit content of the active ingredient contained in a single dose of the medicine of the present invention does not necessarily need to constitute an effective amount, because the necessary effective amount can be achieved by administering multiple dose units.

[0030] The pharmaceutical products of the present invention may also include other active ingredients with similar or different functions. The effective dosage of each active ingredient may vary depending on the ingredients used, the route of administration, and the severity of the condition being treated. Preferably, the total proportion of the various active ingredients administered in the combined formulation may be varied, for example, according to the needs of individual patients with different requirements.

[0031] The beneficial effects of this invention are at least as follows: This invention provides a rapid and efficient method for culturing hypoxia-adapted NK cells in vitro. The NK cells cultured using this method can maintain strong cytotoxic activity and energy metabolism levels even under hypoxic conditions, solving the problem of rapid functional loss of NK cells in the hypoxic tumor microenvironment in existing technologies.

[0032] Compared with methods such as gene modification or drug treatment, the method of the present invention is simple to operate, low in cost, highly stable and reproducible, and easy to promote in laboratory and preclinical studies, and has important scientific and clinical translational significance. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the treatment of each group in Experiment Example 1.

[0035] Figure 2 The in vitro killing activity of NK92 cells against non-small cell lung cancer cells A549 and PC-9 in Experiment Example 1 was measured.

[0036] Figure 3 The results of bulk-seq transcriptome sequencing of NK92 cells in the AH and HPC groups in Experiment Example 2 show the changes in metabolic pathways and ROS detection.

[0037] Figure 4 This is a map showing the detection of NK92 cell function-related markers in the AH and HPC groups in Experiment Example 3.

[0038] Figure 5 The figure shows the statistical results of the detection of NK92 cell function-related markers in the AH group and HPC group in Experiment Example 3. In the figure, ns represents no significant difference.

[0039] Figure 6 The results of in vivo antitumor imaging of NK92 cells in the HPC group in Experiment Example 4.

[0040] Figure 7 The figures show the tumor volume changes after NK92 cell treatment in the HPC group in Experiment Example 4. The first row of the figures shows the average tumor volume changes in each group, while the second and third rows show the tumor volume changes in each experimental animal in each group. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0043] The main experimental materials and methods involved in the specific embodiments of this invention are as follows: 1. Cell lines: A549, PC-9, and 293T cell lines were purchased from the American Type Culture Collection (ATCC), and NK92 cells were purchased from Procell Life Science & Technology. 293T cells were used to culture a stable transgenic strain of A549-Luc virus.

[0044] 2. Cell culture methods: A549 and PC-9 cells were cultured in RPMI 1640 medium: RPMI 1640 (Thermo Fisher Scientific, catalog number 11875119) + 10% fetal bovine serum (ExCell, catalog number FSP500) + penicillin and streptomycin (Thermo Fisher Scientific, catalog number 15140122).

[0045] 293T cells were cultured in DMEM medium (DMEM; Thermo Fisher Scientific, catalog number 11995-065) + 10% fetal bovine serum (ExCell, catalog number FSP500) + penicillin and streptomycin (Thermo Fisher Scientific, catalog number 15140122).

[0046] NK92 cells were cultured in NK92 Cell Complete Medium (Procell Life Science & Technology, catalog number CM-0530).

[0047] All cells were cultured in a humidified cell culture incubator at 37 ℃ and 5% CO2, and mycoplasma testing was performed periodically.

[0048] 3. Mice: Male and female M-NSG mice aged 6-8 weeks (catalog number NM-NSG-001) were purchased from Southern Model Biotechnology Co., Ltd. All male and female mice used in the experiment were bred and maintained at the Guangzhou National Laboratory, with a balanced sex ratio throughout the experiment. The age range of the mice throughout the experiment was 8-18 weeks. Animals were housed under specific pathogen (SPF) conditions, and were housed together with control group animals. At the end of the experiment, mice were euthanized by cervical dislocation after isoflurane anesthesia.

[0049] 4. Construction of the in vivo xenograft model (Cell Line-Derived Xenograft Mode): A549-Luc cells (A549 cells with bioluminescent tags, 5 × 10⁻⁶) 6 Mice were cultured at 37 °C and 5% CO2, and then collected and resuspended in 100 μL of DPBS (Dulbecco's PBS, Thermo Fisher, #14190250) for subcutaneous tumor inoculation. Tumor burden in mice was assessed using in vivo bioluminescence imaging (BLI), and mice were divided into balanced groups based on BLI intensity. Seven days after tumor inoculation, mice were injected via tail vein with 200 μL of NK92 cells resuspended in DPBS (1 × 10⁻⁶ cells). 7 Mice were injected with IL-2 every three days for three consecutive days to maintain NK92 cell activity. Simultaneously, to maintain NK92 cell activity in mice, 10,000 IU of IL-2 was injected every 2-3 days (the specific activity of the IL-2 used in this experiment was 20,000 IU / μg, and 1 IU is equivalent to 0.05 ng IL-2. Based on this, each injection of 10,000 IU IL-2 corresponds to approximately 0.5 μg of protein), for 21 days. Tumor burden was measured using digital calipers, and BLI imaging was performed on representative animals at key stages of tumor growth using the Xenogen IVIS imaging system. Mouse weight and tumor size were recorded and measured every two days. Tumor volume was measured periodically using digital calipers and calculated using the following formula (unit: mm). 3 ): V = L × (W) 2 × 0.5. Where L is the longest diameter of the tumor, and W is the diameter perpendicular to L. Euthanasia was performed when the experiment ended (the tumor reached its maximum permissible volume or the mouse lost its ability to move).

[0050] 5. Functional testing (degranulation and cytokine production): Effector cells (purified NK92) were co-cultured with A549 cells at a 2:1 E:T ratio. After 4 hours of co-culture, the supernatant NK cells were collected and labeled with the following antibodies: • PE-tagged anti-CD107a (RRID:AB_1186062, clone H4A3, BioLegend); • APC-tagged anti-FasL (RRID:AB_2922541, clone NOK-1, BioLegend); • PE-tagged anti-CD69 (RRID:AB_3083391, clone W19310K, BioLegend). • PE-tagged anti-NKG2D (RRID:AB_492961, clone 1D11, BioLegend). • APC-tagged anti-NKG2A (RRID:AB_2888862, clone S19004C, BioLegend); • APC-tagged anti-NKp46 (RRID:AB_2888734, clone 9E2, BioLegend); • PerCP / Cyanine5.5-tagged anti-T-bet (RRID:AB_1595593, clone 4B10, BioLegend); • Brilliant Violet 421-labeled anti-TRAIL (RRID:AB_3683318, clone RIK-2, BioLegend).

[0051] After incubation, cells were stained for viability using Live / Dead Fixable Aqua staining reagent (Thermo Fisher, L-34966) and fixed with 4% paraformaldehyde. Membrane permeation was then performed using Triton X-100, followed by intracellular staining with BV650-labeled anti-T-Bet antibody (RRID: AB_2563608, clone 4S.B3, BioLegend). Cell expression was analyzed using an Agilent NovoCyte™ flow cytometer.

[0052] 6. ROS detection: To detect reactive oxygen species (ROS) levels, 500,000 purified NK cells were collected for each condition and stained with 5 μM MitoSOX Red Mitochondrial Superoxide Indicator (MCE, catalog no. HY-D1055). After staining, the cells were stored on ice and immediately analyzed using Agilent NovoCyte. TM The cells were analyzed using a flow cytometer.

[0053] 7. IncuCyte cytotoxicity assay: A549 and PC-9 tumor cells stably expressing td-tomato and luciferase (A549-td-tomato-luc and PC-9-td-tomato-luc) were seeded into 96-well plates at 8000 cells per well and allowed to adhere overnight. The following day, effector cells NK92 (32000 cells / well) were added to each well, maintaining an effector-to-target cell ratio of 4:1 (E:T = 4:1). The plates were placed in an IncuCyte live-cell imaging system, and fluorescence images of A549 cells were acquired every 0.5 hours, while those of PC-9 cells were acquired every 1.5 hours. All experiments were independently repeated at least three times. Tumor cell cytotoxicity was assessed by quantifying the red cell area (representing td-tomato signal).

[0054] 8. Hypoxic Treatment of NK92 Cells: NK92 cells were subjected to hypoxia treatment using the Huayi Ningchuang Cell+100 intelligent cell preparation system. The oxygen concentrations were set at 1% (upper limit 3%, lower limit 0%), 3% (upper limit 5%, lower limit 1%), and 5% (upper limit 7%, lower limit 3%), while the carbon dioxide concentration was 5%. The system automatically monitored and maintained a stable gas environment throughout the culture process to ensure consistent and reliable hypoxia treatment conditions.

[0055] 9. Detection of cellular ATP production (ATP Assay) Intracellular ATP content was detected using an enhanced ATP assay kit (Beyotime, S0027). Treated cells were collected and lysed, and the supernatant was collected by centrifugation. The cell lysate was mixed with the ATP assay working solution according to the kit instructions, and the luminescence intensity was measured using a chemiluminescence detector. The luminescence signal intensity was positively correlated with the ATP content in the sample, reflecting changes in cellular energy metabolism levels under different treatment conditions.

[0056] 10. Bulk RNA sequencing method (RNA-seq analysis) NK92 cells from the WT, AH, and HPC groups were collected, and total RNA was extracted. RNA quality was assessed, and samples with RNA integrity meeting sequencing requirements were selected for library construction. Transcriptome data were acquired using a high-throughput sequencing platform, and the raw sequencing data underwent quality control, alignment, and standardization. Based on this, differential expression analysis was performed on samples from the HPC and AH groups, and samples from the AH and WT groups. Pathway enrichment and clustering analyses were conducted based on differentially expressed genes. Simultaneously, differential gene expression heatmaps were created for the WT, AH, and HPC groups to visually demonstrate changes in related pathways and gene expression patterns under different culture conditions.

[0057] 11. Statistical analysis: All analyses of differences were performed using SPSS software (version 29.0.1.0). Independent samples t-tests were first performed on the data; when differences were statistically significant (P ≤ 0.05), the corresponding p-values ​​were labeled on the graphs. For comparisons with no significant differences (P > 0.05), the values ​​were labeled as ns on the graphs. All statistical graphs were generated using GraphPad Prism software.

[0058] Example 1 This embodiment provides a method for inducing NK92 cells to tolerate hypoxia in vitro, as detailed below: NK92 cells were cultured under normoxic conditions (21% O2) until the logarithmic growth phase; then cultured sequentially under 5% O2 for 2 days, 3% O2 ​​for 2 days, and 1% O2 for 2 days to complete the hypoxia-induced treatment and obtain hypoxia-adapted NK92 cells.

[0059] The cell culture medium for each stage was NK92 Cell Complete Medium, and the culture temperature was 37℃. The medium was changed every 48 hours.

[0060] Comparative Example 1 This comparative example provides a method for inducing NK92 cells to tolerate hypoxia in vitro, as detailed below: NK92 cells were cultured under normoxic conditions (21% O2) until the logarithmic growth phase; then cultured under 5% O2 for 6 days to complete the hypoxia-induced treatment.

[0061] The cell culture medium for each stage was NK92 Cell Complete Medium, and the culture temperature was 37℃. The medium was changed every 48 hours.

[0062] Comparative Example 2 This comparative example provides a method for inducing NK92 cells to tolerate hypoxia in vitro, as detailed below: NK92 cells were cultured under normoxic conditions (21% O2) until the logarithmic growth phase; then cultured under 1% O2 for 6 days to complete the hypoxia-induced treatment.

[0063] The cell culture medium for each stage was NK92 Cell Complete Medium, and the culture temperature was 37℃. The medium was changed every 48 hours.

[0064] Experimental Example 1 To evaluate the effect of hypoxia adaptation (HPC) on NK cell effector function, in this experiment, hypoxia-adapted NK92 cells obtained from Example 1 (HPC group), Comparative Example 1 (5% group), and Comparative Example 2 (1% group) were first cultured at 21% O2 for 48 h, and then stimulated at 1% O2 for 36 h to simulate the acute hypoxia inhibition faced by NK cells as they enter the tumor microenvironment from the peripheral circulation.

[0065] Wild-type NK cells without 1% O2 conditioned stimulation were set up as the control group (WT), and wild-type NK cells were directly conditioned with 1% O2 for 36 hours as the acute hypoxia control group (AH). See the diagram for the treatment of each group. Figure 1 .

[0066] Subsequently, the in vitro killing activity of NK92 cells against non-small cell lung cancer cells A549 and PC-9 was evaluated using a live-cell real-time imaging system (E:T = 4:1). Results are shown below. Figure 2 The results showed that NK92 cells in the HPC group treated with 1% O2 for 36 hours exhibited target cell-specific cytotoxicity comparable to that of the wild-type control group (WT) cultured under normoxic conditions, and significantly higher than the acute hypoxia control group (AH) treated directly under 1% O2 for 36 hours without hypoxia acclimation. In multiple independent experiments, the killing efficiency of the HPC group was approximately 3-5 times higher than that of the AH group, indicating that hypoxia acclimation can significantly enhance the ability of NK cells to resist acute hypoxia inhibition. Furthermore, compared with hypoxia-treated groups obtained by culture under single constant hypoxia conditions (such as 1% or 5% O2) (Comparative Examples 1 and 2), HPC cells constructed using a gradient hypoxia exposure strategy exhibited superior cytotoxic activity.

[0067] Experiment Example 2 Based on Experiment 1, this experiment further selected NK92 cells from the AH group and HPC group that had been treated with 1% oxygen for 36 hours for bulk RNA sequencing analysis, and simultaneously detected the level of mitochondrial reactive oxygen species (ROS) and intracellular ATP content.

[0068] The results are as follows Figure 3 As shown, compared with the AH group, the expression of genes related to oxidative phosphorylation in NK92 cells of the HPC group showed an overall upward trend, while the expression level of genes related to glycolysis was relatively downward. This suggests that HPC training can reshape the energy metabolism pattern of NK cells under hypoxic conditions, transforming them from an inefficient energy-producing state dominated by glycolysis to a metabolic state dominated by oxidative phosphorylation.

[0069] Consistent with the transcriptome results, functional assays showed that mitochondrial ROS production in the HPC group was significantly lower than that in the AH group, decreasing by approximately two-fold, while intracellular ATP levels were significantly higher, increasing by approximately two-fold. These results indicate that HPC training can improve mitochondrial function and energy metabolism efficiency of NK cells under hypoxic conditions, thus providing a metabolic basis for maintaining their function.

[0070] Experimental Example 3 This experiment further analyzed the functional markers of NK92 cells from the AH and HPC groups treated with 1% oxygen for 36 hours in Experiment 1. The results are shown below. Figure 4 and Figure 5 The results showed that, compared with the AH group, the expression levels of activating markers (CD107a, FasL, NKG2D, NKp46, CD69, T-bet, TRAIL) in the HPC group increased by about 2 to 3 times, while the expression level of the inhibitory marker (NKG2A) decreased by about 2 times.

[0071] Experiment Example 4 This experiment further investigated the antitumor effect of NK92 cells in the HPC group from Experiment 1 in vivo. In vivo imaging was used to monitor tumor growth in NSG mice. The specific method is described above; the main procedure was as follows: each mouse was subcutaneously inoculated with 5 × 10⁶ A549-Luc cells. 6 Seven days later, the cells were injected with either the normoxic group (WT) or the hypoxia-adapted group (HPC) NK92 cells described in Experiment 1, at a dose of 1 × 10⁶ cells per injection. 7 Each animal was injected once every three days, for a total of three injections. Representative animal imaging results during key stages of tumor growth are shown below. Figure 6 The results of tumor size statistics are shown in [the original text]. Figure 7 The study showed that at the end of the observation period, the tumor volume of the HPC group mice was about one-third to one-quarter of that of the WT group, indicating that hypoxia adaptation treatment can effectively reduce the inhibitory effect of hypoxia in the tumor microenvironment on the killing function of NK92 cells.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing NK cells, characterized in that, NK cells were subjected to multiple domestication cultures, with the oxygen concentration decreasing in a gradient during each domestication culture, and the number of domestication cultures was greater than 1.

2. The method for culturing NK cells according to claim 1, characterized in that, The domestication and cultivation process is repeated 3-5 times, with each domestication and cultivation session lasting 2-3 days.

3. The method for culturing NK cells according to claim 1 or 2, characterized in that, The oxygen concentration gradient is reduced by 2-3% during each domestication and cultivation.

4. The method for culturing NK cells according to any one of claims 1-3, characterized in that, When the acclimatization and cultivation are carried out three times, the oxygen concentration during the first acclimatization and cultivation is 5%-6%.

5. The method for culturing NK cells according to any one of claims 1-4, characterized in that, The acclimatization and cultivation process was conducted three times. The oxygen concentration during the first acclimatization and cultivation was 5%, and the oxygen concentration was gradually reduced by 2% during each subsequent acclimatization and cultivation. Each acclimatization and cultivation period lasted for two days.

6. The method for culturing NK cells according to any one of claims 1-5, characterized in that, Before being domesticated, the NK cells were cultured to the logarithmic growth phase under conditions of 20%-22% oxygen concentration.

7. The method for culturing NK cells according to any one of claims 1-6, characterized in that, The NK cells mentioned are NK92 cells.

8. An NK cell, characterized in that, Obtained by culturing NK cells according to any one of claims 1-7.

9. The use of the NK cells according to claim 8 in the preparation of antitumor products.

10. An anti-tumor product, characterized in that, Includes the NK cells as described in claim 8, preferably, the tumor is non-small cell lung cancer.

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