A method for obtaining natural killer cells from adult blood cultures

Abstract

The application relates to the technical field of natural killer cell culture, in particular to a method for obtaining natural killer cells from adult blood culture, which comprises the following steps: obtaining peripheral blood mononuclear cells from blood separation; inoculating the peripheral blood mononuclear cells into an activation culture medium, adding porous microcarriers into the activation culture medium, carrying out suspension culture, and obtaining an activated cell-microcarrier mixture; transferring the activated cell-microcarrier mixture into a culture container, settling to form a fixed bed composed of activated cells and porous microcarriers, then continuously supplementing an expansion culture medium into the culture container in a continuous perfusion mode, discharging culture supernatant containing metabolic by-products, carrying out expansion culture, obtaining expanded cells, and adding polyphenol antioxidants and L-carnitine into the expansion culture medium before the end of the culture, continuing to culture, and harvesting natural killer cells. The application adopts a specific culture strategy, realizes high-fold expansion of NK cells, and makes the obtained NK cells have high killing activity.

Description

Technical Field

[0001] This application relates to the field of natural killer cell culture technology, and more specifically, to a method for obtaining natural killer cells from adult blood culture. Background Technology

[0002] Natural killer (NK) cells are core effector cells in the human innate immune system and belong to a subset of lymphocytes. Unlike T cells and B cells, NK cells do not express specific antigen recognition receptors and can recognize and kill target cells without prior antigen sensitization. Their killing activity is not limited by the major histocompatibility complex (MHC). NK cells exert their anti-tumor and antiviral functions through various mechanisms, including the release of perforin and granzymes, the expression of death receptor ligands, and the secretion of cytokines, serving as the body's first line of defense in immune surveillance. In recent years, the rise of engineered NK cell therapies such as chimeric antigen receptor NK cells (CAR-NK) and the safety advantage of NK cells in allogeneic transplantation, which is less likely to induce graft-versus-host disease, have made NK cell-based immunotherapy a research hotspot in the field of tumor cell therapy. However, the content of NK cells in human peripheral blood is limited, accounting for only 5% to 20% of peripheral blood lymphocytes, and their in vivo lifespan is relatively short. Therefore, establishing efficient and stable in vitro expansion methods to obtain sufficient quantities and quality of NK cells is a key prerequisite for promoting the clinical application of NK cells.

[0003] Currently, the in vitro culture methods for NK cells generally employ static culture systems, which use culture flasks or bags to culture cells in a quiescent state, with fresh culture medium added intermittently. In this static intermittent feeding method, byproducts such as lactic acid produced by cell metabolism continuously accumulate during the feeding intervals. Accumulated lactic acid not only lowers the pH of the culture medium but also directly inhibits the proliferative activity and cytotoxic function of NK cells, becoming a key factor limiting NK cell expansion efficiency and functional maintenance. Summary of the Invention

[0004] The purpose of this application is to overcome the aforementioned shortcomings of the prior art and provide a method for obtaining natural killer (NK) cells from adult blood culture. This method employs a specific culture strategy to achieve high-multiplication of NK cells while simultaneously ensuring that the obtained NK cells possess high cytotoxic activity.

[0005] To achieve the above objectives, this application provides the following technical solution: A method for obtaining natural killer cells from adult blood culture includes the following steps: Peripheral blood mononuclear cells were obtained from blood. The peripheral blood mononuclear cells were seeded in an activation culture medium containing porous microcarriers, the surface of which was coupled with CD16 antibody and NKG2D ligand. Suspension culture was performed to obtain a mixture of activated cells and microcarriers; The mixture of activated cells and microcarriers is transferred to a culture container and allowed to settle to form a fixed bed of activated cells and porous microcarriers. Then, amplification culture medium is added to the culture container by continuous perfusion, and the culture supernatant containing metabolic byproducts is discharged. Perform amplification culture to obtain expanded cells; Before the amplification culture ends, polyphenolic antioxidants and L-carnitine are added to the amplification culture medium. After culturing for a longer period, natural killer cells are harvested.

[0006] In the technical solution of this application, the entire culture process is set into three continuous stages: the microcarrier suspension activation stage, the static perfusion amplification stage, and the metabolic enhancement maturation stage. The microcarrier suspension activation stage corresponds to the steps from cell seeding to obtaining a mixture of activated cells and microcarriers; the static perfusion amplification stage corresponds to the steps of transferring the mixture to a culture container, allowing it to settle to form a fixed bed, and then performing continuous perfusion amplification; the metabolic enhancement maturation stage corresponds to the steps of adding polyphenolic antioxidants and L-carnitine before the end of the amplification culture and continuing the culture.

[0007] In the microcarrier suspension activation stage, porous microcarriers with surface-conjugated CD16 antibody and NKG2D ligand were used as the supporting medium for cell activation and growth. The microcarriers provided cells with a three-dimensional attachment surface, and their porous structure further increased the surface area available for cell attachment, while also facilitating the inward diffusion of nutrients and oxygen, thus improving mass transfer efficiency. The CD16 antibody on the microcarrier surface could bind to the CD16 receptor (FcγRIII) on the surface of NK cells, mimicking the activation signal of antibody-dependent cell-mediated cytotoxicity; the NKG2D ligand could bind to the NKG2D receptor on the surface of NK cells, providing a co-stimulatory signal. These two activation signals functioned through different signal transduction pathways. The CD16-mediated ADCC effector signaling pathway and the NKG2D-mediated co-stimulatory signaling pathway synergistically worked together with the three-dimensional culture space to ensure sufficient activation of NK cells while preventing premature depletion.

[0008] In the static perfusion expansion phase, the activated cells and microcarrier mixture are transferred to a culture vessel and allowed to settle to form a fixed bed. After the microcarrier particles settle, the gaps between the particles form perfusion channels, allowing the culture medium to flow uniformly within the particle gaps without generating excessive shear forces. Fresh expansion medium is then continuously added via continuous perfusion, while the culture supernatant containing metabolic byproducts is simultaneously removed. This phase utilizes the stable, low-shear environment provided by the microcarrier fixed bed, combined with continuous perfusion to supply nutrients and remove metabolic inhibitory factors such as lactate, enabling NK cells to expand under conditions unaffected by contact inhibition and metabolite accumulation.

[0009] During the metabolic enhancement and maturation phase, a short-term treatment with polyphenolic antioxidants and L-carnitine was added to the culture medium before the end of the expansion culture. Polyphenolic antioxidants activate the AMPK signaling pathway and promote mitochondrial biosynthesis; L-carnitine, as a fatty acid transporter, promotes the entry of long-chain fatty acids into mitochondria for β-oxidation. The synergistic effect of these two agents allows the expanded NK cells to accumulate more functional mitochondria. This increase in mitochondria provides NK cells with more ATP energy support, thereby enhancing their granzyme reserves and degranulation ability, resulting in high cytotoxic activity at harvest.

[0010] The synergistic combination of these three culture stages achieves a balance between high-expansion and high cytotoxic activity of NK cells. Three-dimensional activation via microcarriers provides a sufficient yet gentle initiation signal, avoiding premature depletion caused by continuous stimulation from high concentrations of soluble cytokines; perfusion culture removes metabolic byproducts, eliminating the metabolic inhibition bottleneck during expansion; and pre-harvest metabolic enhancement treatment compensates for potential mitochondrial functional dilution caused by high-expansion. This sequential combination of the three stages results in NK cells with both high expansion rates and high cytotoxic activity.

[0011] Furthermore, the culture vessel is a perfusion bioreactor. A perfusion bioreactor can precisely control the perfusion rate, temperature, and gas exchange, providing a stable culture environment for the cells.

[0012] The activation medium is a basal medium containing mesenchymal stem cell conditioned medium and IL-15. The mesenchymal stem cell conditioned medium contains various paracrine factors that promote NK cell survival and early activation; while IL-15 is a key cytokine for NK cell development and survival. The two work synergistically to provide mild and sustained activation support. Furthermore, in the activation medium, the volume fraction of the mesenchymal stem cell conditioned medium is 10%–30%, and the concentration of IL-15 is 1–10 ng / mL.

[0013] The porous microcarriers are made of polylactic-co-glycolic acid copolymer or dextran. These materials exhibit good biocompatibility and controllable degradation. The microcarrier particle size is 100–300 μm, a range that provides a sufficiently large specific surface area for cell attachment while ensuring the formation of a fixed bed with good perfusion channels after static settling. The CD16 antibody and NKG2D ligand are covalently coupled to the microcarrier surface. Compared to physical adsorption coating, covalent coupling ensures the stable presence of the activating factors during perfusion culture.

[0014] The NKG2D ligand is either MICA protein (MHC class I chain-related protein A) or ULBP protein (UL16-binding protein). Both MICA and ULBP are natural ligands of the NKG2D receptor in the human body and can effectively activate the NKG2D signaling pathway.

[0015] The suspension culture conditions were 37°C and 5%... The culture time is 24–48 hours. This time range allows NK cells to receive sufficient activation signals while avoiding premature exhaustion of effector functions due to excessively long activation time.

[0016] The amplification medium is a basal medium containing IL-2 and IL-21. IL-2 is a classic stimulatory factor for NK cell proliferation; IL-21 can promote the differentiation and functional maturation of NK cells. Further, in the amplification medium, the concentration of IL-2 is 50-200 IU / mL, and the concentration of IL-21 is 10-50 ng / mL.

[0017] During the expansion culture process, the lactate concentration in the culture medium was maintained below 2 g / L through perfusion. Maintaining a low lactate concentration effectively relieves the inhibitory effect of lactate on NK cell proliferation and cytotoxicity. The total expansion culture time was 7–14 days, within which NK cells could achieve the clinically required dose-like expansion.

[0018] The polyphenolic antioxidant is resveratrol or pterostilbene, added at a concentration of 5–20 μM. The L-carnitine is added at a concentration of 0.5–2 mM. Adding these two substances to the culture medium 12–48 hours before the end of the amplification culture allows for effective enhancement of cell function without significantly affecting the amplification process.

[0019] Compared with the prior art, this application has the following advantages: By coupling CD16 antibody and NKG2D ligand to the surface of microcarriers, a synergistic activation signal is provided. Combined with the three-dimensional culture space provided by the three-dimensional microcarriers, efficient and gentle activation of NK cells is achieved, avoiding the problem of premature depletion caused by traditional high-concentration cytokine stimulation.

[0020] By employing a continuous perfusion culture method to continuously remove metabolic byproducts, the bottleneck of NK cell proliferation inhibition by the accumulation of metabolic byproducts under static intermittent feeding was overcome, thereby increasing the expansion rate.

[0021] Before harvest, polyphenolic antioxidants and L-carnitine were introduced for short-term metabolic enhancement treatment to compensate for the dilution of mitochondrial function that may be caused by high-fold expansion, so that the obtained NK cells have both high expansion fold and high killing activity. Detailed Implementation

[0022] To enable those skilled in the art to better understand this application, the technical solutions of this application are described in detail below through specific embodiments. The reagents and raw materials used in the following embodiments, comparative examples, and experimental examples are all commercially available products, and the experimental methods used, unless otherwise specified, are conventional methods in the art.

[0023] The following detailed explanation of specific implementation methods further illustrates this application: Example 1

[0024] This embodiment provides a method for obtaining natural killer cells from adult blood culture, including the following steps: S100. Obtaining peripheral blood mononuclear cells from blood: Collect 50 mL of adult peripheral blood, dilute with an equal volume of PBS buffer, and slowly add to the upper layer of Ficoll separation solution. Centrifuge at 700 g for 20 minutes, aspirate the middle white membrane layer, and wash twice with PBS to obtain peripheral blood mononuclear cells. Purified NK cells are obtained from the peripheral blood mononuclear cells using an NK cell sorting kit (Miltenyi, Human NK Cell Isolation Kit).

[0025] S200. Porous microcarriers were prepared by weighing polylactic-co-glycolic acid copolymer (PLGA, molecular weight 50,000) and using a double emulsion solvent evaporation method. Microcarriers with a particle size of 150–250 μm were collected by sieving. The microcarriers were immersed in a coupling buffer containing 1 mg / mL CD16 monoclonal antibody and 1 mg / mL MICA protein. Carbodiimide and N-hydroxysuccinimide were added for covalent coupling, and the reaction was carried out overnight at 4°C. After the reaction, the microcarriers were thoroughly washed with PBS to obtain porous microcarriers with CD16 antibody and NKG2D ligand coupled to their surface. The protein coupling amount per milliliter of microcarrier was determined to be approximately 80–120 μg.

[0026] Preparation of S300, activation medium and amplification medium: Activation medium: Add 20% mesenchymal stem cell conditioned medium and IL-15 to serum-free RPMI-1640 basal medium.

[0027] Amplification medium: IL-2 with a final concentration of 100 IU / mL and IL-21 with a final concentration of 30 ng / mL were added to serum-free RPMI-1640 basal medium.

[0028] The method for preparing the conditioned medium for mesenchymal stem cells is as follows: human umbilical cord mesenchymal stem cells (commercially available products, or obtained by isolation and culture from umbilical cord tissue according to conventional methods in this field) are taken and cultured. The cells were seeded at a density of cells / cm² in culture flasks and cultured in DMEM medium containing 10% fetal bovine serum until 80% confluence. The old medium was then discarded, and the cells were washed twice with PBS. The medium was then replaced with serum-free RPMI-1640 basal medium and cultured for another 48 hours. The culture supernatant was collected, centrifuged at 1000g for 10 minutes to remove cell debris, and filtered through a 0.22μm filter membrane to obtain the conditioned medium for mesenchymal stem cells.

[0029] It should be noted that the conditioned medium for mesenchymal stem cells prepared above has been replaced with serum-free RPMI-1640 basal medium during the preparation process.

[0030] S400, The purified NK cells obtained in step S100 are then... The microcarriers were inoculated at a density of 10 / mL into the activation medium, which contained porous microcarriers prepared in step S200 (microcarrier volume accounting for 10% of the medium volume). The culture was then incubated at 37°C with a 5% concentration of [missing information]. The cells were cultured in suspension for 48 hours in an incubator, during which they were continuously shaken at 50 rpm to obtain a mixture of activated cells and microcarriers.

[0031] S500: Transfer the mixture of activated cells and microcarriers obtained in step S400 into a perfusion bioreactor. Stop shaking and allow the microcarriers and attached cells to settle naturally, forming a fixed bed with a height of approximately 2 cm. At this time, the cell density in the fixed bed is approximately... Cells / mL (based on fixed bed volume). Day 0 of the expansion culture was defined as the start of perfusion. Expansion culture medium was continuously added to the reactor at a perfusion rate of 0.3 mL / min, while the culture supernatant containing metabolic byproducts was simultaneously discharged from the top of the reactor at the same rate. During the expansion culture, lactate concentration in the culture medium was sampled every 24 hours, and the lactate concentration was maintained below 2 g / L by adjusting the perfusion rate. The expansion culture lasted for 10 days (ending on day 10), yielding expanded cells.

[0032] S600. On the 9th day of amplification culture (i.e., 24 hours before the end of amplification culture), add resveratrol to the amplification medium to a final concentration of 10 μM and add L-carnitine to a final concentration of 1 mM. After continuing perfusion culture for 24 hours, harvest natural killer cells. Example

[0033] The difference between this embodiment and Example 1 is that the porous microcarrier in step S200 is made of dextran, the perfusion rate in step S500 is 0.1 mL / min, and resveratrol and L-carnitine are added on day 9.5 of the amplification culture (i.e., 12 hours before the end of the amplification culture) in step S600. The remaining steps are the same as in Example 1. Example

[0034] The difference between this embodiment and Example 1 is that the NKG2D ligand in step S200 is ULBP protein, the perfusion rate in step S500 is 0.5 mL / min, the amplification culture in step S500 lasts for 14 days, and in step S600, pterostilbene (final concentration 20 μM) and L-carnitine (final concentration 2 mM) are added on day 12 of the amplification culture (i.e., 48 hours before the end of the amplification culture). The remaining steps are the same as in Example 1.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that in step S200, the porous microcarrier is only coupled with the CD16 antibody and not with the NKG2D ligand. The remaining steps are the same as in Example 1.

[0036] Comparative Example 2 The difference between this comparative example and Example 1 is that in step S500, instead of continuous perfusion, an intermittent feeding method is used, that is, half the volume of old culture medium is aspirated every 2 days and an equal volume of fresh amplification culture medium is added. The remaining steps are the same as in Example 1.

[0037] Comparative Example 3 The difference between this comparative example and Example 1 is that resveratrol and L-carnitine are not added in step S600; instead, cells are harvested directly. The remaining steps are the same as in Example 1.

[0038] Comparative Example 4 The difference between this comparative example and Example 1 is that the static perfusion amplification stage of step S500 is omitted. Specifically, the mixture of activated cells and microcarriers obtained in step S400 is not transferred to the perfusion bioreactor, but to a conventional cell culture flask. Static culture is used, and half the volume of old culture medium is aspirated every two days, and an equal volume of fresh amplification culture medium is added. The remaining steps are the same as in Example 1.

[0039] Comparative Example 5 The difference between this comparative example and Example 1 is that no protein is coupled to the surface of the porous microcarrier in step S200; only unmodified bare porous microcarriers are used. The remaining steps are the same as in Example 1.

[0040] Comparative Example 6 The difference between this comparative example and Example 1 is that, in step S400, porous microcarriers are not used. Instead, CD16 antibody and NKG2D ligand are added directly to the activation medium in soluble form, with a final concentration of 10 μg / mL for both CD16 antibody and MICA protein. This amount of soluble addition corresponds to the total amount of protein conjugated to the microcarriers per milliliter of medium in Example 1.

[0041] It should be noted that, since there are no microcarriers for forming a fixed bed in this comparative example, the static sedimentation process to form a fixed bed is not performed in step S500. Instead, the activated cell suspension is directly transferred to the perfusion bioreactor, and the amplification culture medium is continuously added to the reactor in a suspension perfusion manner. The culture supernatant containing metabolic byproducts is discharged from the top of the reactor at the same rate. The remaining steps are the same as in Example 1.

[0042] Experimental Example 1 Cell proliferation fold and cytotoxic activity assay: Cell suspensions from Examples 1-3 and Comparative Examples 1-6 were collected before harvest (i.e., at the end of amplification culture), and the number of viable cells was counted using trypan blue staining. The amplification fold was calculated using the following formula:

[0043] Simultaneously, NK cells harvested from Examples 1-3 and Comparative Examples 1-6 were used as effector cells, and K562 tumor cells were used as target cells. LDH release assay was performed to detect cytotoxicity at an effector-to-target ratio of 10:1. Effector cells and target cells were added to 96-well plates and incubated at 37°C for 5%...

[0044] After 4 hours of incubation, the LDH activity in the culture supernatant was detected, and the kill rate was calculated using the following formula. :

[0045] In the formula, The OD value of the experimental group. The OD value of the spontaneous release group of effector cells. The OD value of the target cell spontaneous release group. This represents the OD value of the target cell maximum release group.

[0046] The results are shown in the table below (the data in the table are the average of three independent experiments): Example 1 Approximately 850 times Approximately 78% Example 2 Approximately 720 times Approximately 74% Example 3 Approximately 910 times Approximately 81% Comparative Example 1 Approximately 480 times Approximately 62% Comparative Example 2 Approximately 520 times Approximately 65% Comparative Example 3 Approximately 830 times Approximately 58% Comparative Example 4 Approximately 350 times Approximately 56% Comparative Example 5 Approximately 390 times Approximately 59% Comparative Example 6 Approximately 510 times Approximately 63% As can be seen from the table above, the amplification fold and killing activity of Examples 1-3 are significantly higher than those of the respective pairs.

[0047] Regarding the amplification fold, Comparative Example 1 showed a significantly lower amplification fold due to the lack of a co-activating signal from the NKG2D ligand, indicating that the dual conjugation of CD16 antibody and NKG2D ligand has a significant synergistic promoting effect on NK cell amplification. Comparative Example 5 used a bare microcarrier, and the amplification fold was similar to that of Comparative Example 1, further demonstrating that although the microcarrier itself provides a three-dimensional attachment surface, efficient amplification cannot be achieved without the conjugated activating factor. Comparative Example 6 directly added the activating factor in a soluble form, and the amplification fold was also significantly lower than that of the examples, indicating that covalently conjugating the CD16 antibody and NKG2D ligand to the surface of the microcarrier provides a more sustained and effective activation signal compared to adding it in a soluble form. Comparative Example 2, by using an intermittent feeding method instead of continuous perfusion, saw the accumulation of metabolic byproducts inhibit NK cell proliferation, resulting in a significantly lower amplification fold than the examples using the continuous perfusion method, indicating that continuous perfusion plays an important role in maintaining the amplification environment. Comparative Example 4, which completely omitted the perfusion amplification stage and only used conventional static culture, showed a further significant decrease in the amplification fold, indicating that the amplification method of microcarrier fixed bed combined with continuous perfusion has significant advantages over conventional static culture.

[0048] Regarding cytotoxic activity, Comparative Example 3, lacking pre-harvest metabolic enhancement treatment, exhibited the lowest cytotoxic activity, indicating that pre-harvest metabolic enhancement with polyphenolic antioxidants and L-carnitine plays a crucial role in maintaining and enhancing NK cell cytotoxic activity. Comparative Example 5 showed similar cytotoxic activity to Comparative Example 3, suggesting that while the naked microcarrier can support some cell growth, insufficient activation also affects the cytotoxic function of the final product. Comparative Example 6 showed lower cytotoxic activity than the examples, indicating that the soluble activating factors added were easily diluted under perfusion conditions, failing to provide a sustained activation signal and leading to decreased cell cytotoxic activity. Comparative Example 4, omitting the perfusion amplification stage, showed a low amplification fold and unsatisfactory cytotoxic activity, further confirming the importance of continuous perfusion and control of metabolic byproducts in maintaining cell function.

[0049] Experimental Example 2 Purity detection of NK cells, the final product of culture: Cells harvested from Example 1, Comparative Example 1, Comparative Example 5, and Comparative Example 6 were labeled with CD3-FITC antibody and CD56-PE antibody, and then analyzed by flow cytometry. Cell ratio.

[0050] The results showed that the cells harvested in Example 1 The proportion was approximately 96%, compared to approximately 89% in comparative example 1, 82% in comparative example 5, and 85% in comparative example 6. This indicates that the NKG2D ligand coupled to the surface of the microcarrier has a selective promoting effect on the specific amplification of NK cells, while the addition of activating factors to the bare microcarrier or in soluble form cannot effectively enrich NK cells.

[0051] Experimental Example 3 Validation of the time window for metabolic enhancement treatment: Following the method in Example 1, resveratrol and L-carnitine were added at different time points (6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours) before the end of the amplification culture for metabolic enhancement treatment. After harvest, the NK cell killing activity (effect-to-target ratio 10:1) was detected.

[0052] The results showed that the kill rate was approximately 72% after 12 hours of treatment, approximately 78% after 24 hours, approximately 80% after 48 hours, and approximately 81% after 72 hours. The kill rate after 6 hours of treatment was approximately 60%, showing only a limited improvement compared to control group 3 (approximately 58%) without metabolic enhancement treatment. These results indicate that a metabolic enhancement treatment time window of 12–48 hours can significantly enhance NK cell killing activity, while treatment times of less than 12 hours have little effect.

[0053] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the embodiments above are only for the purpose of helping to understand the core ideas of this application. It should be noted that those skilled in the art can make several improvements and modifications to this application without departing from the principles of this application, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A method for obtaining natural killer cells from adult blood culture, characterized in that: Peripheral blood mononuclear cells were obtained from blood. The peripheral blood mononuclear cells were seeded in an activation culture medium containing porous microcarriers, the surface of which was coupled with CD16 antibody and NKG2D ligand. Suspension culture was performed to obtain a mixture of activated cells and microcarriers; The mixture of activated cells and microcarriers is transferred to a culture container and allowed to settle to form a fixed bed of activated cells and porous microcarriers. Then, amplification culture medium is added to the culture container by continuous perfusion, and the culture supernatant containing metabolic byproducts is discharged. Perform amplification culture to obtain expanded cells; Before the amplification culture ends, polyphenolic antioxidants and L-carnitine are added to the amplification culture medium. After culturing for a longer period, natural killer cells are harvested.

2. The method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The NKG2D ligand is either MICA protein or ULBP protein.

3. The method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The porous microcarrier is made of polylactic acid-glycolic acid copolymer or dextran, with a particle size of 100-300 μm; the CD16 antibody and NKG2D ligand are covalently coupled to the surface of the porous microcarrier.

4. The method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The amplification culture time is 7 to 14 days, and the lactate concentration in the culture medium is maintained below 2 g / L by perfusion throughout the amplification culture period.

5. The method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The polyphenolic antioxidant and the L-carnitine are added to the amplification culture medium at a time that is 12 to 48 hours before the end of the amplification culture.

6. The method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The polyphenolic antioxidant is resveratrol or pterostilbene, with an addition concentration of 5–20 μM; the addition concentration of L-carnitine is 0.5–2 mM.

7. The method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The activation culture medium is a basal culture medium containing mesenchymal stem cell conditioned medium and IL-15; The mesenchymal stem cell conditioned medium has a volume fraction of 10% to 30%, and the concentration of IL-15 is 1 to 10 ng / mL.

8. A method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The amplification medium is a basal medium containing IL-2 and IL-21; The concentration of IL-2 is 50–200 IU / mL, and the concentration of IL-21 is 10–50 ng / mL.

9. A method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The culture vessel is a perfusion bioreactor.

10. A method for obtaining natural killer cells from adult blood culture according to claim 1, characterized in that: The suspension culture conditions were 37°C and 5%... The incubation period is 24 to 48 hours.

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