In-vitro culture medium for efficiently amplifying NK cells, culture method and application thereof
By optimizing NK cell culture conditions with serum-free culture medium and specific additives, the safety and efficiency issues of NK cell expansion were resolved, achieving stable expansion of high-purity, highly active NK cells suitable for clinical applications.
Patent Information
- Application Number
- CN202511952233.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-01-27
AI Technical Summary
Existing NK cell expansion technologies have safety risks, unstable expansion efficiency, and cell purity and functional activity that are difficult to meet clinical needs. They are also prone to T cell contamination and in vivo activity attenuation.
Serum-free culture medium combined with pyruvate, glutamine, IL-15, IL-21 and the phosphate ester small molecule activator SY001, combined with a hypoxic environment and specific additives such as astragalus polysaccharide, berberine alkaloids and Ganoderma triterpenes, was used to optimize culture conditions to promote NK cell expansion.
It achieves efficient and stable NK cell expansion with high cell purity and long-lasting functional activity, reducing production costs and making it suitable for large-scale clinical applications.
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Figure CN121406576A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to an in vitro culture medium, culture method and application for high-efficiency expansion of NK cells. Background Technology
[0002] Natural killer (NK) cells, as core effector units of the innate immune system, play a dual role in anti-tumor and anti-infective immune responses. These cells can directly recognize and eliminate tumor cells, virus-infected cells, and dysfunctional cells without prior antigen sensitization. Simultaneously, NK cells, by secreting cytokines such as interferon-γ and tumor necrosis factor-α, can effectively activate adaptive immune cells such as T cells and dendritic cells, forming a complete anti-tumor immune regulatory network. Recent clinical studies have confirmed that NK cells have made groundbreaking progress in the treatment of solid tumors, particularly demonstrating unique advantages in the treatment of melanoma, colorectal cancer, and other solid tumors.
[0003] However, NK cells only account for 5-10% of peripheral blood lymphocytes, limiting their direct clinical application. Therefore, establishing an efficient in vitro expansion system has become a core technical aspect of NK cell therapy. Currently, the mainstream expansion strategies are mainly divided into two categories: feeder cell culture systems and antibody-cytokine combination culture systems. Among them, while genetically modified K562 cells can achieve thousand-fold expansion of NK cells as feeder cells, this approach has significant safety risks. It requires gamma-ray irradiation treatment to eliminate tumorigenic risks, and residual radiation-resistant cells may cause safety issues such as insertional mutations. On the other hand, the use of antibody-cytokine complexes for expansion avoids the biosafety risks associated with cell contact, but it generally suffers from unstable expansion efficiency and high cell phenotypic heterogeneity, making it difficult to meet the stringent standards for NK cell purity and functional activity required in clinical treatment.
[0004] Currently, existing culture systems struggle to maintain the natural killing properties of NK cells while ensuring high cell survival rates. Furthermore, large-scale expansion is prone to contamination by non-target cells such as T cells, affecting treatment safety. In contrast, NK cells obtained through traditional methods exhibit hyperfunction in vitro and are easily depleted after reinfusion, leading to a decline in their anti-tumor activity in vivo.
[0005] Therefore, developing a serum-free culture system that combines high expansion efficiency, high cell purity, and high functional durability to overcome the key technological barriers restricting the clinical translation of NK cells has become a major scientific problem that urgently needs to be solved in the field of tumor immunotherapy. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing an efficient in vitro culture medium for expanding NK cells, a culture method therefor, and its application.
[0007] A highly efficient in vitro culture medium for expanding natural killer cells, using serum-free medium as the base medium, further comprising: pyruvate, glutamine, IL-15, IL-21, and the phosphate ester small molecule activator SY001; wherein the concentration of pyruvate in the in vitro culture medium is 1-5 mmol / L, the concentration of glutamine in the in vitro culture medium is 2-4 mmol / L, the concentration of IL-15 in the in vitro culture medium is 10-50 ng / mL, the concentration of IL-21 in the in vitro culture medium is 5-20 ng / mL, and the concentration of the phosphate ester small molecule activator SY001 in the in vitro culture medium is 0.1-1 μmol / L.
[0008] The serum-free culture medium can be the NK cell serum-free culture medium produced by Hangzhou Zhongying Biomedical Technology Co., Ltd., catalog number: ZY-NKJ-1000.
[0009] The structural formula of SY001, a small molecule activator of phosphate esters, is as follows: , It can mimic cytokine signal transduction.
[0010] Specifically, the phosphate ester small molecule activator SY001 can be prepared using the following steps: (1) Using anhydrous dichloromethane (DCM) as solvent, and phenol derivatives (such as methyl paraben) and phosphorus oxychloride (POCl3) as raw materials; phosphorus oxychloride was added dropwise to the phenol derivatives under ice bath conditions, and the reaction was stirred for 2 hours. During the stirring process, the temperature was slowly restored to room temperature; then phosphorus oxychloride was removed by vacuum distillation to obtain a white solid [Ar-OP(O)Cl2] (yield ≥85%). The above reaction equation is: Ar-OH + POCl3 → Ar-OP(O)Cl2 + HCl; (2) Using tetrahydrofuran (THF) as solvent, Ar-OP(O)Cl2 obtained in step (1) and 2-pyrrolidineethanol were used as raw materials. The two were reacted at room temperature for 4 hours. Triethylamine was added dropwise during the reaction to neutralize the generated HCl. Triethylamine hydrochloride (Et3N·HCl) was removed by filtration and then purified by column chromatography (silica gel, DCM / MeOH=10:1) to obtain a colorless oily substance, namely the phosphate ester small molecule activator SY001 (yield 75%). The above reaction equation is: Ar-OP(O)Cl2 + HO-(CH2)2-NR2 → Ar-OP(O)-O-(CH2)2-NR2 +HCl.
[0011] Preferably, the in vitro culture medium further includes: Astragalus polysaccharide; wherein the concentration of Astragalus polysaccharide in the in vitro culture medium is 0.5-1.2 mg / mL.
[0012] Preferably, the in vitro culture medium further includes: berberine and ganoderic triterpenes; wherein the berberine concentration in the in vitro culture medium is 1-3 μmol / L, and the final concentration of ganoderic triterpenes is 0.1-0.2 mg / mL.
[0013] A highly efficient in vitro culture method for expanding natural killer cells includes the following steps: isolating peripheral blood mononuclear cells (PBMCs) from healthy donors and collecting CD56+CD3- NK cells; seeding the NK cells in 6-well plates coated with anti-CD16 antibody at a density of 1–10 × 10⁻⁶ cells / well. 5 cells / mL; NK cells were cultured in a hypoxic environment using the above-mentioned in vitro culture medium for at least 14 days, with fresh in vitro culture medium added every 48 hours.
[0014] Preferably, the temperature of the low-oxygen environment is 36-38℃; the volume fraction of each gas in the low-oxygen environment is as follows: oxygen 5%, carbon dioxide 5%, and the balance is nitrogen.
[0015] Preferably, half of the culture medium is replaced to replenish the above-mentioned in vitro culture medium with fresh medium.
[0016] A highly efficient in vitro culture method for expanding natural killer (NK) cells includes the following steps: isolating peripheral blood mononuclear cells (PBMCs) from healthy donors and collecting CD56+CD3- NK cells; seeding NK cells in 6-well plates coated with anti-CD16 antibody at a density of 1–10 × 10⁵ cells / mL; culturing NK cells in the above-mentioned in vitro culture medium under hypoxic conditions for 4 days, supplementing with fresh in vitro culture medium (containing 0.5–1.2 mg / mL Astragalus polysaccharide) every 48 hours; culturing NK cells again in the above-mentioned in vitro culture medium under hypoxic conditions for 4 days, supplementing with fresh in vitro culture medium (containing 1–3 μmol / L Coptis chinensis alkaloids and 0.1–0.2 mg / mL Ganoderma lucidum triterpenes) every 48 hours; and then culturing NK cells in the above-mentioned in vitro culture medium under hypoxic conditions for at least 6 days, supplementing with fresh in vitro culture medium (without Astragalus polysaccharide, Coptis chinensis alkaloids, and Ganoderma lucidum triterpenes) every 48 hours.
[0017] Preferably, the temperature of the low-oxygen environment is 36-38℃; the volume fraction of each gas in the low-oxygen environment is as follows: oxygen 5%, carbon dioxide 5%, and the balance is nitrogen.
[0018] Preferably, half of the culture medium is replaced to replenish the above-mentioned in vitro culture medium with fresh medium.
[0019] An application of expanded NK cells in the preparation of products for treating hematologic malignancies or solid tumors, wherein the expanded NK cells are obtained by the above-mentioned in vitro culture method.
[0020] Beneficial effects: 1. This invention uses serum-free culture medium combined with autologous cytokine combination (IL-15 / IL-21), eliminating the use of gene-modified trophoblast cells and eliminating the risk of tumorigenic residue from the source; furthermore, astragalus polysaccharide is added to the serum-free culture medium from 0-4 days, which can effectively induce early activation of NK cells and promote rapid expansion of initial NK cells. Combined with the addition of berberine and Ganoderma triterpenes from 5-8 days, the secretion of cytotoxic granules is enhanced, the natural cytotoxic reserve is maintained, and the killing activity of NK cells against tumor cells is comprehensively enhanced.
[0021] 2. The present invention adds the phosphate ester small molecule activator SY001 to enhance signal transduction by targeting NK cell surface receptors (such as NKp30), forming a positive feedback loop with IL-15 / IL-21, synergistically improving amplification efficiency and cytotoxicity, breaking through the limitation of cell doubling efficiency decreasing with each generation in traditional amplification systems, and achieving steady-state amplification.
[0022] 3. This invention does not use feeder cells, effectively avoiding ethical controversies and pollution risks, while extending the in vitro survival time of NK cells by optimizing energy metabolism.
[0023] 4. Experiments have shown that the NK cell expansion of this invention reaches more than 1550 times, the proportion of CD3-CD56+ cells is >90%, and the killing activity against K562 cells (effect-to-target ratio 1:1) is increased to more than 85%.
[0024] 5. This invention greatly saves production costs, is easy to operate, and has high production efficiency, making it fully applicable to large-scale clinical research. Attached Figure Description
[0025] Figure 1 This is a comparison chart of cell survival rate and cytotoxic activity of NK cells after in vitro culture using the methods of Example 1 and Comparative Example 1.
[0026] Figure 2 The graph shows a comparison of the expansion efficiency of NK cells and the proportion of CD107a cells after using the in vitro culture methods of Examples 1-2 and Comparative Examples 1-3.
[0027] Figure 3 The graph shows a comparison of IFN-γ secretion and glycolysis rate of NK cells after in vitro culture using the methods described in Examples 1-2 and Comparative Examples 1-3.
[0028] Figure 4The graph shows a comparison of tumor volume on day 28 and survival rate on day 60 in mice from the control group, comparative example 1 group, example 1 group, and example 2 group.
[0029] Figure 5 This is a comparison of the number of NK cells surviving in mice in Comparative Example 1, Example 1, and Example 2 groups 7 days after infusion.
[0030] Figure 6 The graph shows the fold increase of NK cells, the proportion of CD3-CD56+ cells, and the comparison of cell viability after cryopreservation and thawing following the in vitro culture methods of Examples 1 and 2. Detailed Implementation
[0031] The present invention will be further explained below with reference to specific embodiments.
[0032] Example 1: A highly efficient in vitro culture method for expanding natural killer cells includes the following steps: (1) PBMCs were isolated from peripheral blood of healthy donors and CD56+CD3- NK cells were sorted by magnetic beads; (2) NK cells were seeded in 6-well plates coated with anti-CD16 antibody at a density of 2 × 10⁶ cells / well. 5 cells / mL; (3) The serum-free NK cell culture medium (Hangzhou Zhongying Biomedical Technology Co., Ltd., catalog number: ZY-NKJ-1000) was used as the basic culture medium, and pyruvate, glutamine, IL-15, IL-21 and phosphate ester small molecule activator SY001 were added to obtain the first in vitro culture medium; the final concentration of pyruvate in the system was 3 mmol / L, the final concentration of glutamine in the system was 3 mmol / L, the final concentration of IL-15 in the system was 30 ng / mL, the final concentration of IL-21 in the system was 10 ng / mL, and the final concentration of phosphate ester small molecule activator SY001 in the system was 0.5 μmol / L; NK cells were cultured in a hypoxic environment (37°C, 5% O2, 5% CO2) using the above-mentioned in vitro culture medium for 14 days, with half of the above-mentioned in vitro culture medium being replaced every 48 hours.
[0033] The cultured system was tested and found that the NK cell expansion reached 1550-fold, the proportion of CD3-CD56+ cells was >90%, and the killing activity against K562 cells (effect-to-target ratio 1:1) was increased to 85%.
[0034] Comparative Example 1: NK cells were cultured using the traditional IL-2 amplification method as a positive control (cultured for 14 days). Upon completion of the culture, the NK cell amplification was only 10-fold.
[0035] Cell viability and cytotoxic activity were tested in the systems cultured in Example 1 and Comparative Example 1. Figure 1 As shown, the in vitro culture method of Example 1 can significantly improve cell survival rate and killing activity.
[0036] Comparative Example 2: A highly efficient in vitro culture method for expanding natural killer cells includes the following steps: (1) PBMCs were isolated from peripheral blood of healthy donors and CD56+CD3- NK cells were sorted by magnetic beads; (2) NK cells were seeded in 6-well plates coated with anti-CD16 antibody at a density of 2 × 10⁶ cells / well. 5 cells / mL; (3) The serum-free NK cell culture medium (Hangzhou Zhongying Biomedical Technology Co., Ltd., catalog number: ZY-NKJ-1000) was used as the basic culture medium, and IL-15 and IL-21 were added to obtain the first in vitro culture medium; the final concentration of IL-15 in the system was 30 ng / mL, and the final concentration of IL-21 in the system was 10 ng / mL. NK cells were cultured in a hypoxic environment (37°C, 5% O2, 5% CO2) using the above-mentioned in vitro culture medium for 14 days, with half of the above-mentioned in vitro culture medium being replaced every 48 hours.
[0037] Comparative Example 3: A highly efficient in vitro culture method for expanding natural killer cells includes the following steps: (1) PBMCs were isolated from peripheral blood of healthy donors and CD56+CD3- NK cells were sorted by magnetic beads; (2) NK cells were seeded in 6-well plates coated with anti-CD16 antibody at a density of 2 × 10⁶ cells / well. 5 cells / mL; (3) The serum-free NK cell culture medium (Hangzhou Zhongying Biomedical Technology Co., Ltd., catalog number: ZY-NKJ-1000) was used as the basic culture medium, and IL-15, IL-21 and phosphate ester small molecule activator SY001 were added to obtain the first in vitro culture medium; the final concentration of IL-15 in the system was 30 ng / mL, the final concentration of IL-21 in the system was 10 ng / mL, and the final concentration of phosphate ester small molecule activator SY001 in the system was 0.5 μmol / L; NK cells were cultured in a hypoxic environment (37°C, 5% O2, 5% CO2) using the above-mentioned in vitro culture medium for 14 days, with half of the above-mentioned in vitro culture medium being replaced every 48 hours.
[0038] Example 2: A highly efficient in vitro culture method for expanding natural killer cells includes the following steps: (1) PBMCs were isolated from peripheral blood of healthy donors and CD56+CD3- NK cells were sorted by magnetic beads; (2) NK cells were seeded in 6-well plates coated with anti-CD16 antibody at a density of 2 × 10⁶ cells / well. 5 cells / mL; (3) The serum-free NK cell culture medium (Hangzhou Zhongying Biomedical Technology Co., Ltd., catalog number: ZY-NKJ-1000) was used as the basic culture medium, and pyruvate, glutamine, IL-15, IL-21 and phosphate ester small molecule activator SY001 were added to obtain the first in vitro culture medium; the final concentration of pyruvate in the system was 3 mmol / L, the final concentration of glutamine in the system was 3 mmol / L, the final concentration of IL-15 in the system was 30 ng / mL, the final concentration of IL-21 in the system was 10 ng / mL, and the final concentration of phosphate ester small molecule activator SY001 in the system was 0.5 μmol / L; Using serum-free NK cell culture medium (Hangzhou Zhongying Biomedical Technology Co., Ltd., catalog number: ZY-NKJ-1000) as the basal medium, a second in vitro culture medium was prepared by adding pyruvate, glutamine, IL-15, IL-21, the phosphate ester small molecule activator SY001, and astragalus polysaccharide. The final concentrations of pyruvate, glutamine, IL-15, IL-21, SY001, and astragalus polysaccharide were 3 mmol / L, 30 ng / mL, 10 ng / mL, 0.5 μmol / L, and 0.8 mg / mL, respectively. Using serum-free NK cell culture medium (Hangzhou Zhongying Biomedical Technology Co., Ltd., catalog number: ZY-NKJ-1000) as the basal medium, a third in vitro culture medium was prepared by adding pyruvate, glutamine, IL-15, IL-21, the phosphate ester small molecule activator SY001, berberine, and Ganoderma triterpenes. The final concentrations of pyruvate, glutamine, IL-15, IL-21, SY001, 2 μmol / L, 2 μmol / L, and 0.15 mg / mL were determined. NK cells were cultured in a hypoxic environment using a second in vitro culture medium for 4 days, with half of the medium being replaced every 48 hours. Then, NK cells were cultured in a hypoxic environment using a third in vitro culture medium for 4 days, with half of the medium being replaced every 48 hours. Finally, NK cells were cultured in a hypoxic environment using a first in vitro culture medium for at least 6 days, with half of the medium being replaced every 48 hours.
[0039] The amplification efficiency, functional analysis, metabolic status, and gene expression of the culture systems from Examples 1-2 and Comparative Examples 1-3 were determined. Details are as follows: (1) Amplification efficiency: Cell count and viability in each group were determined by trypan blue staining; (2) Functional analysis: Flow cytometry detection of CD107a (degranulation marker) and IFN-γ secretion (ELISA); (3) Metabolic state: Seahorse analyzed glycolysis rate (ECAR) and mitochondrial respiration (OCR); (4) Gene expression: RNA-seq analysis of mTOR pathway-related genes (such as AKT and S6K).
[0040] Results for each group are as follows Figure 2 and Figure 3 As shown.
[0041] Compared with Comparative Example 2, Comparative Example 3 showed that the phosphate ester small molecule activator SY001 significantly increased the amplification rate, activated the mTORC1 pathway (RNA-seq analysis of mTOR pathway-related genes showed a 2.5-fold increase in S6K phosphorylation level), and promoted NK cell proliferation. This indicates that the phosphate ester small molecule activator SY001 can enhance signal transduction by targeting NK cell surface receptors (such as NKp30) and synergize with IL-15 / IL-21 (not simply additively).
[0042] Compared with Comparative Example 3, Example 1 showed that pyruvate / glutamine supplementation increased the glycolysis rate to 95%. Meanwhile, mitochondrial respiration (OCR) analysis showed that the mitochondrial OCR of Example 1 increased by 1.8 times, indicating that the metabolic reprogramming was a "glycolysis-oxidative phosphorylation coupled" mode, avoiding cell apoptosis caused by lactate accumulation. That is, through metabolic optimization, NK cells can be stably expanded in vitro for ≥14 days (survival rate >95%), which is suitable for large-scale production.
[0043] Compared to Comparative Examples 1-2, the in vitro culture methods of Examples 1 or 2 significantly increased the cell proliferation rate, the proportion of CD107a+ cells, IFN-γ secretion, and glycolysis rate. While the in vitro culture method of Example 2 significantly increased the cell proliferation rate, the proportion of CD107a+ cells, and IFN-γ secretion compared to Example 1, there was no significant difference in glycolysis rate between the two methods. This confirms that the combined use of Astragalus polysaccharides, Coptis chinensis alkaloids, and Ganoderma lucidum triterpenes can further promote NK cell proliferation.
[0044] Humanized PDX mouse models (lung cancer xenografts) were used, and mice were randomly divided into 4 groups. The control group received PBS; the control group received NK cells (1×10⁻⁶) expanded from the control group. 6 cells / mouse); Example 1 group was infused with NK cells obtained from the expansion in Example 1 (1×10 cells / mouse); Example 1 group was infused with NK cells obtained from Example 1 expansion (1×10 cells / mouse). 6 cells / mouse); Example 2 group was infused with NK cells obtained from the expansion in Example 2 (1×10 cells / mouse); Example 2 group was infused with NK cells obtained from Example 2 expansion (1×10 cells / mouse). 6 (cells / mouse).
[0045] The result is as follows Figure 4 and Figure 5 As shown, the tumor volume in Example 1 and Example 2 groups was significantly smaller than that in the control group and Comparative Example 1 group, and the survival rate and in vivo NK cell survival count were significantly higher than those in the control group and Comparative Example 1 group. Compared with Example 1 group, the tumor volume in Example 2 group was even smaller, and the survival rate and in vivo NK cell survival count were even greater. This confirms that the combined use of Astragalus polysaccharide, Coptis chinensis alkaloids, and Ganoderma lucidum triterpenes enhances the secretion of cytotoxic granules, maintains the natural cytotoxic reserve, and comprehensively enhances the killing activity of NK cells against tumor cells.
[0046] Meanwhile, NK cells in both Example 1 and Example 2 groups highly expressed the homing receptor CXCR4 (over 80% as detected by flow cytometry, far exceeding the 30% of the control group), promoting enrichment at the tumor site; and no cytokine storm was observed (serum IL-6 and TNF-α levels were not different from the control group).
[0047] The expansion consistency of three batches of NK cells from different donor sources was tested in a GMP-grade bioreactor. Figure 6 As shown, the method of the present invention can stably expand functional NK cells (RSD < 5%), meeting the needs of clinical-grade production.
[0048] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A highly efficient in vitro culture medium for expanding natural killer cells, characterized in that, The serum-free culture medium is the base medium, which also includes: pyruvate, glutamine, IL-15, IL-21 and phosphate ester small molecule activator SY001.
2. The in vitro culture medium according to claim 1, characterized in that, The concentration of pyruvate in the in vitro culture medium is 1-5 mmol / L, the concentration of glutamine in the in vitro culture medium is 2-4 mmol / L, the concentration of IL-15 in the in vitro culture medium is 10-50 ng / mL, the concentration of IL-21 in the in vitro culture medium is 5-20 ng / mL, and the concentration of the phosphate ester small molecule activator SY001 in the in vitro culture medium is 0.1-1 μmol / L.
3. The in vitro culture medium according to claim 1, characterized in that, The in vitro culture medium further includes: Astragalus polysaccharide; wherein the concentration of Astragalus polysaccharide in the in vitro culture medium is 0.5-1.2 mg / mL; Preferably, the in vitro culture medium further includes: berberine and ganoderic triterpenes; wherein the concentration of berberine in the in vitro culture medium is 1-3 μmol / L, and the concentration of ganoderic triterpenes in the in vitro culture medium is 0.1-0.2 mg / mL.
4. A method for efficiently expanding natural killer cells in vitro, characterized in that, The procedure includes the following steps: isolating PBMCs from healthy donor peripheral blood and collecting CD56+CD3- NK cells; seeding the NK cells in 6-well plates coated with anti-CD16 antibody at a density of 1–10 × 10⁻⁶ cells / well. 5 cells / mL; NK cells were cultured in a hypoxic environment for at least 14 days using the in vitro culture medium of claim 1, wherein fresh in vitro culture medium of claim 1 was added every 48 hours.
5. The in vitro culture method according to claim 4, characterized in that, The temperature of the low-oxygen environment is 36-38℃; the volume fraction of each gas in the low-oxygen environment is as follows: oxygen 5%, carbon dioxide 5%, and the balance is nitrogen.
6. The in vitro culture method according to claim 4, characterized in that, The in vitro culture medium of claim 1 is replenished by half-volume replacement.
7. A method for efficiently expanding natural killer cells in vitro, characterized in that, The procedure includes the following steps: isolating PBMCs from healthy donor peripheral blood and collecting CD56+CD3- NK cells; seeding the NK cells in 6-well plates coated with anti-CD16 antibody at a density of 1–10 × 10⁻⁶ cells / well. 5 cells / mL; NK cells were cultured in a hypoxic environment for 4 days using the in vitro culture medium of claim 2, and fresh in vitro culture medium of claim 2 was added every 48 hours; NK cells were then cultured in a hypoxic environment for 4 days using the in vitro culture medium of claim 2, and fresh in vitro culture medium of claim 3 was added every 48 hours; NK cells were then cultured in a hypoxic environment for at least 6 days using the in vitro culture medium of claim 1, and fresh in vitro culture medium of claim 1 was added every 48 hours.
8. The in vitro culture method according to claim 7, characterized in that, The temperature of the low-oxygen environment is 36-38℃; the volume fraction of each gas in the low-oxygen environment is as follows: oxygen 5%, carbon dioxide 5%, and the balance is nitrogen.
9. The in vitro culture method according to claim 7, characterized in that, The in vitro culture medium described in claim 1, 2, or 3 is replenished by half-volume replacement.
10. The application of expanded NK cells in the preparation of products for treating hematologic malignancies or solid tumors, characterized in that, The NK cells were amplified using the in vitro culture method described in any one of claims 4-9.