A method for culturing induced nk cells

Abstract

The application discloses a culture method of induced NK cells in the NK field, and starts separation from peripheral blood mononuclear cells, establishes an induction culture system and a co-culture system, uses an induction culture solution prepared from natural plant extracts and lactobacillus, provides the cells with biologically active components, can effectively promote the growth, differentiation or induction of specific immune responses of the cells, and can simulate the cell interaction environment in the body by co-culturing human nephroblastoma cells with peripheral blood mononuclear cells, and researches the interaction between cells or the response of cells to the induction culture solution.

Description

Technical Field

[0001] This invention belongs to the field of NK cell culture technology, specifically referring to a method for culturing induced NK cells. Background Technology

[0002] Natural killer (NK) cells are an important component of the body's innate immune system, capable of exerting anti-tumor and antiviral effects by recognizing and killing virus-infected or tumor cells. Unlike T cells, NK cells do not require activation by specific antigens but instead spontaneously kill target cells through the response of their surface receptors. Therefore, NK cells have broad application prospects in immunotherapy, tumor immunotherapy, and viral infection control.

[0003] However, the number of NK cells in vivo is limited, and their application is somewhat restricted due to the difficulties in isolation, proliferation, and functional maintenance during in vitro culture. To overcome this challenge, different activation methods and culture conditions have been used to promote the large-scale expansion of NK cells from peripheral blood in vitro and enhance their cytotoxic activity. Improvements in NK cell culture methods have not only promoted basic research on NK cells but also provided more possibilities for clinical treatment.

[0004] NK cells are a type of lymphocyte, mainly divided into two subtypes: CD56*bright and CD56*dim. CD56*bright NK cells are mainly distributed in lymphoid organs and have a strong cytokine secretion capacity; while CD56*dim NK cells are more common in peripheral blood and have a strong killing function. The function of NK cells mainly depends on the interaction between their surface receptors and target cell surface molecules. NK cells recognize abnormal signals in target cells through their activation receptors (e.g., lack of major histocompatibility complex class I molecules (MHC-I) or expression of stress-inducing molecules) and kill target cells by releasing cytotoxic molecules (such as granzymes and perforin).

[0005] Traditional NK cell expansion methods typically rely on isolating NK cells from peripheral blood and then culturing them in vitro. However, simple expansion may not effectively enhance NK cell function. Cell isolation and culture medium selection are crucial: The first step in NK cell culture is to isolate monocytes from peripheral blood or umbilical cord blood, and then obtain purified NK cells using sorting techniques (such as magnetic bead sorting, flow cytometry, etc.). NK cell isolation is often achieved through the difference in surface markers CD56 and CD3. To obtain more efficient NK cell expansion, the choice of culture medium is critical. Commonly used media include RPMI-1640 and IMDM, which typically contain fetal bovine serum (FBS) to provide necessary growth factors. Cytokine use is also important: NK cell culture requires not only suitable culture media but also specific cytokines to enhance NK cell proliferation and activity. Commonly used cytokines include IL-2, IL-15, and IL-21. IL-2 is a classic NK cell growth factor that significantly promotes NK cell proliferation. IL-15 promotes NK cell self-renewal and proliferation, and maintains their functional activity. IL-21 enhances the cytotoxicity and cytokine secretion of NK cells and is often used to increase their anti-tumor activity. Activation and Expansion: In addition to using cytokines, scientists have also attempted to enhance the killing function of NK cells through activation during expansion. For example, using monoclonal antibodies to activate NK cell activation receptors (such as NKG2D and NKp46), or activating small molecule compounds or natural ligands (such as IL-12 and IL-18), can effectively improve NK cell activity. During cell culture, feeder cells, such as K562 cells, can be added. These feeder cells provide essential signals to promote NK cell proliferation and activation. During expansion, the quality of NK cells needs to be monitored regularly, including testing cell purity, activity, and killing function. Commonly used detection methods include flow cytometry (detecting cell surface markers), cell proliferation assays, and cytotoxicity assays (such as 51Cr release assays or apoptosis rate measured by flow cytometry). These assays help researchers evaluate the effectiveness of culture methods and ensure that the cultured NK cells have high anti-tumor function. Summary of the Invention

[0006] To address the above issues and overcome the shortcomings of existing technologies, this invention provides a method for culturing induced NK cells. This invention begins with the isolation of peripheral blood mononuclear cells, establishing an induction culture system and a co-culture system. An induction culture medium prepared using natural plant extracts (Astragalus membranaceus, Eucommia ulmoides, and beetroot) and probiotics (Lactobacillus plantarum) provides the cells with bioactive components that effectively promote cell growth, differentiation, or induce specific immune responses. By co-culturing human nephroblastoma cells with peripheral blood mononuclear cells, the in vivo cell-cell interaction environment can be simulated, allowing for the study of cell-cell interactions or cell responses to the induction culture medium.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention proposes a method for culturing induced NK cells, specifically including the following steps:

[0008] S1: Isolation of peripheral blood mononuclear cells:

[0009] S11. Take peripheral blood from the antecubital vein of a healthy person, add heparin sodium for anticoagulation, centrifuge the whole blood system at room temperature at 700-800g for 10 minutes, then set the speed to 1 to separate cells and plasma. The upper layer is the plasma layer and the lower layer is the blood cell layer. Take the upper plasma layer, place it in a centrifuge tube, seal it, and place it in a water bath for inactivation.

[0010] S12. After inactivation, the plasma was placed at 4℃ for 30 minutes and then centrifuged at 900-1000g for 10 minutes at room temperature with an elevation of 8 and a descent of 5. The upper plasma layer was transferred to a new centrifuge tube and stored at 4℃ to obtain autologous serum. 0.9% physiological saline was injected into the lower plasma layer and mixed evenly to obtain a blood cell suspension.

[0011] S13. Take the blood cell suspension described in step S12 and inject it into 10-20 mL of lymphocyte separation medium. Centrifuge at 700-800 g for 20 min at room temperature, with a 2-fold increase and 1-fold decrease. Transfer the mononuclear cell layer to a new centrifuge tube, add 0.9% physiological saline, mix well, and centrifuge at 700-800 g for 10 min at room temperature, with a 9-fold increase and 9-fold decrease. Discard the supernatant, add 0.9% physiological saline to resuspend the cells, repeat the centrifugation steps, discard the supernatant, add RPMI-1650 medium to prepare a cell suspension, and store it at 4°C for later use.

[0012] S2: Preparation of induction culture medium:

[0013] S21. Astragalus membranaceus and Eucommia ulmoides were pulverized separately to obtain Astragalus membranaceus powder and Eucommia ulmoides powder respectively. The Astragalus membranaceus powder and Eucommia ulmoides powder were mixed and added to a round bottom flask. Purified water was added and the temperature was raised to 70-100℃ for water bath heating and reflux reaction. After reacting for 1-4 hours, the mixture was filtered, the filtrate was collected, concentrated to 1 / 3 of the original volume, and ethanol solution was added for alcohol precipitation. After standing overnight, the mixture was filtered, the precipitate was collected, and the complex polysaccharide was obtained.

[0014] Preferably, in step S21, the mass ratio of Astragalus powder to Eucommia powder is 3-5:1;

[0015] S22. After washing the beets, peel and cut them into pieces. Place them in a low-speed juicer and juice them at a speed of 50-80 rpm. After filtering, collect the filtrate to obtain beet juice.

[0016] S23. Activate *Lactobacillus plantarum*, spread it on MRS agar plates, pick a single colony and place it in MRS broth medium. Incubate at 37°C for 24 hours to obtain a bacterial suspension with a viable cell concentration of 1.0 × 10⁻⁶. 8 -5.0×10 8 CFU / mL;

[0017] S24. Mix the beet juice prepared in step S2 and the complex polysaccharide prepared in step S21, add the bacterial solution prepared in step S23 at a volume fraction of 1%-5%, place it at 33-38℃ and ferment for 18-36 hours. Then, centrifuge the fermentation broth at 4℃ at a speed of 6000-8000 rpm for 5 minutes, raise the temperature to 90℃ for sterilization for 0.5-1 hour, cool it, and store it at 4℃ for later use to obtain the induction culture medium.

[0018] Preferably, in step S24, the mass concentration of the complex polysaccharide in the beet juice is 0.05-0.15 g / mL;

[0019] S3: Cell Culture

[0020] S31. Human nephroblastoma cell lines were cultured in McCoy's 5A medium containing 15% FBS and 1% P / S, and then neutrally cultured in an incubator at 37°C and 5% CO2. When the cell density reached 70%-90%, trypsin digestion solution containing 0.25% EDTA was added for digestion, and then passaged.

[0021] S32. Dilute the peripheral blood mononuclear cells obtained in step S1 to 1.0 × 10⁶ cells / mL using PRMI-1640 containing 10% FBS, and distribute them at a concentration of 1.0 × 10⁶ cells / mL per well. 3 -2.0×10 3Peripheral blood mononuclear cells were transferred to co-culture chambers at 1 per well.

[0022] S33, human nephroblastoma cells were divided into groups of 1.0 × 10⁻⁶. 3 -2.0×10 3 Seeds were seeded per well in a 6-well plate. A suspended co-culture chamber was placed in each well and incubated at 37°C and 5% CO2. Induction culture medium prepared in step S2 was added to each well at 100 μL, and CD16 antibody was added to each well at 15 μL. The plates were then co-cultured for 24 h.

[0023] The beneficial effects achieved by this invention are as follows:

[0024] This invention provides a method for culturing induced NK cells. The method begins with the isolation of peripheral blood mononuclear cells, establishing an induction culture system and a co-culture system. The induction culture medium prepared using natural plant extracts (Astragalus membranaceus, Eucommia ulmoides, and beet) and probiotics (Lactobacillus plantarum) provides the cells with bioactive components that can effectively promote cell growth, differentiation, or induce specific immune responses. By co-culturing human nephroblastoma cells with peripheral blood mononuclear cells, the in vivo cell interaction environment can be simulated to study cell-cell interactions or cell responses to the induction culture medium. Astragalus and Eucommia ulmoides possess immunomodulatory and antiviral functions. They can promote the secretion of functional factors such as IL-6 and IL-8 by human nephroblastoma cells and induce increased NK cell killing activity. Through the induction of polysaccharides from Astragalus and Eucommia, they can not only act on the proliferation and growth of NK cells but also induce human nephroblastoma cells to secrete related inducible cytokines, thereby enhancing the proliferative performance and killing power of NK cells. Using polysaccharides from beet, Eucommia, and Astragalus as substrates, fermentation with Lactobacillus plantarum can convert large polysaccharides into small short-chain fatty acids, providing energy and substrate support for glycolysis and TCA pathways in the later stages of NK cell activation, thereby improving the proliferative capacity of NK cells in the later stages of activation. Attached Figure Description

[0025] Figure 1 The graph shows the expression levels of various molecules in the NK cells cultured in Examples 1-5.

[0026] Figure 2 The graph shows the results of the IFN-γ release capacity of NK cells cultured in Examples 1-5;

[0027] Figure 3 The graph shows the cytotoxic activity results of K562 target cells of NK cells cultured in Examples 1-5 of this invention.

[0028] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials and test strains used in the following examples were purchased from commercial channels.

[0032] Example 1

[0033] This embodiment provides a method for culturing NK cells, specifically including the following steps:

[0034] S1: Isolation of peripheral blood mononuclear cells:

[0035] S11. Collect 50 mL of peripheral blood from the antecubital vein of a healthy volunteer, add 1 mg of heparin sodium for anticoagulation, mix well, place the whole blood system at room temperature, centrifuge at 800 g for 10 min, separate the upper plasma layer and the lower cell layer, use a Busbar tube to pick up the upper plasma layer and place it in a new sterile centrifuge tube, seal the centrifuge tube with sealing film, and place it in a water bath at 55°C for 30 min for inactivation, and then set aside for use.

[0036] S12. After the inactivated plasma is placed at 4℃ and allowed to stand for 30 minutes, it is centrifuged at 1000g for 10 minutes at room temperature with an elevation of 8 and a descent of 5. The upper plasma layer is transferred to a new sterile centrifuge tube and stored at 4℃ to obtain autologous serum. 0.9% physiological saline is injected into the lower plasma layer and mixed evenly by inverting the tube to obtain a blood cell suspension.

[0037] S13. Take 20 mL of the blood cell suspension prepared in step S12 and centrifuge at 700 g for 20 min at room temperature. Then, transfer the mononuclear cell layer to a new sterile centrifuge tube, add 0.9% physiological saline, mix well, and centrifuge at 800 g for 9 min at room temperature. After discarding the supernatant, add 0.9% physiological saline to resuspend the cells. Repeat the centrifugation step twice, discard the supernatant, add RPMI-1650 medium to prepare a cell suspension, and store it at 4°C for later use.

[0038] S2: Cell Culture

[0039] S21. Add 5 mL of PBS and 15 μL of LCD16 antibody to a T25 culture flask, mix well, and incubate at 37°C in 5% CO2 for 2 hours. Then, aspirate any excess liquid and set aside.

[0040] S22. Take the cell suspension prepared in step S13 and adjust the cell concentration to 2.0 × 10⁻⁶. 6 Add the cells / mL to the T25 culture flask from step S21, mix gently, and then add 10 mL of serum-free human lymphocyte culture medium prepared according to IL-2 50 ng / mL, IL-12 50 ng / mL, IL-15 200 ng / mL, IL-18 100 ng / mL, and IL-21 50 ng / mL. Add this medium to the T25 culture flask, and add the autologous serum prepared in step S12 at 5 vol% of the serum-free human lymphocyte culture medium. Mix gently and thoroughly, and then culture at 37°C and 5% CO2. Count the cells using trypan blue staining.

[0041] Example 2

[0042] This embodiment provides a method for culturing NK cells, specifically including the following steps:

[0043] S1: Isolation of peripheral blood mononuclear cells:

[0044] S11. Collect 50 mL of peripheral blood from the antecubital vein of a healthy volunteer, add 1 mg of heparin sodium for anticoagulation, mix well, place the whole blood system at room temperature, centrifuge at 800 g for 10 min, separate the upper plasma layer and the lower cell layer, use a Busbar tube to pick up the upper plasma layer and place it in a new sterile centrifuge tube, seal the centrifuge tube with sealing film, and place it in a water bath at 55°C for 30 min for inactivation, and then set aside for use.

[0045] S12. After the inactivated plasma is placed at 4℃ and allowed to stand for 30 minutes, it is centrifuged at 1000g for 10 minutes at room temperature with an elevation of 8 and a descent of 5. The upper plasma layer is transferred to a new sterile centrifuge tube and stored at 4℃ to obtain autologous serum. 0.9% physiological saline is injected into the lower plasma layer and mixed evenly by inverting the tube to obtain a blood cell suspension.

[0046] S13. Take 20 mL of the blood cell suspension prepared in step S12 and centrifuge at 700 g for 20 min at room temperature. Then, transfer the mononuclear cell layer to a new sterile centrifuge tube, add 0.9% physiological saline, mix well, and centrifuge at 800 g for 9 min at room temperature. After discarding the supernatant, add 0.9% physiological saline to resuspend the cells. Repeat the centrifugation step twice, discard the supernatant, add RPMI-1650 medium to prepare a cell suspension, and store it at 4°C for later use.

[0047] S2: Bacterial culture:

[0048] S21. Human nephroblastoma cell lines were cultured in McCoy's 5A medium containing 15% FBS and 1% P / S, and then neutrally cultured in an incubator at 37°C and 5% CO2. When the cell density reached 89.3%, a digestion solution containing 0.25% EDTA trypsin was added for digestion, and the cells were then passaged.

[0049] S22. Dilute the peripheral blood mononuclear cells obtained in step S1 to 1.43 × 10⁻⁶ using PRMI-1640 containing 10% FBS. 6 cells / mL, at a rate of 1.84 × 10⁻⁶ cells / well. 3 Peripheral blood mononuclear cells were transferred to co-culture chambers at 1 per well.

[0050] S23, human nephroblastoma cells were divided into groups of 1.53 × 10⁻⁶. 3 Seeds were seeded per well in a 6-well plate. A suspended co-culture chamber was placed into each well and incubated at 37°C and 5% CO2. 15 μL of CD16 antibody was added to each well of the co-culture chamber and the plates were co-cultured for 24 h.

[0051] Example 3

[0052] S1: Isolation of peripheral blood mononuclear cells:

[0053] S11. Collect peripheral blood from the healthy elbow vein, add heparin sodium for anticoagulation, centrifuge the whole blood system at room temperature at 800g for 10 minutes, then set the speed to 1 to separate cells and plasma. The upper layer is the plasma layer and the lower layer is the blood cell layer. Take the upper plasma layer, place it in a centrifuge tube, seal it, and place it in a water bath for inactivation.

[0054] S12. After inactivation, the plasma was placed at 4°C for 30 minutes and then centrifuged at 900g for 10 minutes at room temperature with an elevation of 8 and a descent of 5. The upper plasma layer was transferred to a new centrifuge tube and stored at 4°C to obtain autologous serum. 0.9% physiological saline was injected into the lower plasma layer and mixed evenly to obtain a blood cell suspension.

[0055] S13. Take the blood cell suspension described in step S12 and inject it into 15 mL of lymphocyte separation medium. Centrifuge at 800 for 20 min at room temperature, with a 2-fold increase and 1-fold decrease. Transfer the mononuclear cell layer to a new centrifuge tube, add 0.9% physiological saline, mix well, and centrifuge at 700-800g for 10 min at room temperature, with a 9-fold increase and 9-fold decrease. Discard the supernatant, add 0.9% physiological saline to resuspend the cells, repeat the centrifugation steps, discard the supernatant, add RPMI-1650 medium to prepare a cell suspension, and store it at 4℃ for later use.

[0056] S2: Preparation of induction culture medium:

[0057] S21. Astragalus membranaceus and Eucommia ulmoides were pulverized separately to obtain Astragalus membranaceus powder and Eucommia ulmoides powder respectively. 3g of Astragalus membranaceus powder and 1g of Eucommia ulmoides powder were mixed and added to a round-bottom flask. 120mL of purified water was added and the temperature was raised to 70℃ for water bath heating and reflux reaction. After the reaction was carried out for 4 hours, the mixture was filtered, the filtrate was collected, concentrated to 1 / 3 of the original volume, and ethanol solution was added for alcohol precipitation. After standing overnight, the mixture was filtered, the precipitate was collected, and the complex polysaccharide was obtained.

[0058] S22. After washing the beets, peel and cut them into pieces. Place them in a low-speed juicer and juice them at 50 rpm. After filtering, collect the filtrate to obtain beet juice.

[0059] S23. Activate *Lactobacillus plantarum*, spread it on MRS agar plates, pick a single colony and place it in MRS broth medium. After incubation at 37°C for 24 hours, the bacterial suspension is obtained with a viable cell concentration of 1.21 × 10⁻⁶. 8 CFU / mL;

[0060] S24. Mix 20 mL of beet juice prepared in step S2 and 1 g of complex polysaccharide prepared in step S21, add 5% by volume to the bacterial solution prepared in step S23, place at 38℃ and ferment for 18 h, centrifuge the fermentation broth at 8000 rpm for 5 min at 4℃, then sterilize at 90℃ for 0.5 h, cool, and store at 4℃ for later use to obtain the induction culture medium;

[0061] S3: Cell Culture

[0062] S31. Human nephroblastoma cell lines were cultured in McCoy's 5A medium containing 15% FBS and 1% P / S, and then neutrally cultured in an incubator at 37°C and 5% CO2. When the cell density reached 74.5%, a digestion solution containing 0.25% EDTA trypsin was added for digestion, and the cells were then passaged.

[0063] S32. Dilute the peripheral blood mononuclear cells obtained in step S1 to 1.67 × 10⁻⁶ using PRMI-1640 containing 10% FBS. 6 cells / mL, at a rate of 1.27 × 10⁻⁶ per well. 3 Peripheral blood mononuclear cells were transferred to co-culture chambers at 1 per well.

[0064] S33, human nephroblastoma cells were divided into groups of 1.06 × 10⁻⁶. 3 Seeds were seeded per well in a 6-well plate. A suspended co-culture chamber was placed in each well and incubated at 37°C and 5% CO2. Induction culture medium prepared in step S2 was added to each well at 100 μL, and CD16 antibody was added to each well at 15 μL. The plates were then co-cultured for 24 h.

[0065] Example 4

[0066] S1: Isolation of peripheral blood mononuclear cells:

[0067] S11. Collect peripheral blood from the healthy elbow vein, add heparin sodium for anticoagulation, centrifuge the whole blood system at room temperature at 800g for 10 minutes, then set the speed to 1 to separate cells and plasma. The upper layer is the plasma layer and the lower layer is the blood cell layer. Take the upper plasma layer, place it in a centrifuge tube, seal it, and place it in a water bath for inactivation.

[0068] S12. After inactivation, the plasma was placed at 4°C for 30 minutes and then centrifuged at 900g for 10 minutes at room temperature with an elevation of 8 and a descent of 5. The upper plasma layer was transferred to a new centrifuge tube and stored at 4°C to obtain autologous serum. 0.9% physiological saline was injected into the lower plasma layer and mixed evenly to obtain a blood cell suspension.

[0069] S13. Take the blood cell suspension described in step S12 and inject it into 20 mL of lymphocyte separation medium. Centrifuge at 800 g for 20 min at room temperature, with a 2-degree incline and 1-degree incline. Transfer the mononuclear cell layer to a new centrifuge tube, add 0.9% physiological saline, mix well, and centrifuge at 700 g for 10 min at room temperature, with a 9-degree incline and 9-degree incline. Discard the supernatant, add 0.9% physiological saline to resuspend the cells, repeat the centrifugation steps, discard the supernatant, add RPMI-1650 medium to prepare a cell suspension, and store it at 4°C for later use.

[0070] S2: Preparation of induction culture medium:

[0071] S21. Astragalus membranaceus and Eucommia ulmoides were pulverized separately to obtain Astragalus membranaceus powder and Eucommia ulmoides powder respectively. 4g of Astragalus membranaceus powder and 1g of Eucommia ulmoides powder were mixed and added to a round-bottom flask. 100mL of purified water was added and the temperature was raised to 70℃ for water bath heating and reflux reaction. After the reaction was carried out for 4 hours, the mixture was filtered, the filtrate was collected, concentrated to 1 / 3 of the original volume, and ethanol solution was added for alcohol precipitation. After standing overnight, the mixture was filtered, the precipitate was collected, and the complex polysaccharide was obtained.

[0072] S22. After washing the beets, peel and cut them into pieces. Place them in a low-speed juicer and juice them at 50 rpm. After filtering, collect the filtrate to obtain beet juice.

[0073] S23. Activate *Lactobacillus plantarum*, spread it on MRS agar plates, pick a single colony and place it in MRS broth medium. After incubation at 37°C for 24 hours, the bacterial suspension is obtained with a viable cell concentration of 1.43 × 10⁻⁶. 8 CFU / mL;

[0074] S24. Mix 10 mL of beet juice prepared in step S2 and 1 g of complex polysaccharide prepared in step S21, add 10% by volume to the bacterial solution prepared in step S23, place at 33℃ and ferment for 36 h. After fermentation, centrifuge the fermentation broth at 4℃ at 6000 rpm for 10 min, then sterilize at 90℃ for 1 h, cool, and store at 4℃ for later use to obtain the induction culture medium.

[0075] S3: Cell Culture

[0076] S31. Human nephroblastoma cell lines were cultured in McCoy's 5A medium containing 15% FBS and 1% P / S, and then neutrally cultured in an incubator at 37°C and 5% CO2. When the cell density reached 82.3%, trypsin digestion solution containing 0.25% EDTA was added for digestion, and then passaged.

[0077] S32. Dilute the peripheral blood mononuclear cells obtained in step S1 to 1.86 × 10⁻⁶ using PRMI-1640 containing 10% FBS. 6 cells / mL, at a rate of 1.76 × 10⁻⁶ per well. 3 Peripheral blood mononuclear cells were transferred to co-culture chambers at 1 per well.

[0078] S33, human nephroblastoma cells were divided into groups of 1.45 × 10⁻⁶. 3Seeds were seeded per well in a 6-well plate. A suspended co-culture chamber was placed in each well and incubated at 37°C and 5% CO2. Induction culture medium prepared in step S2 was added to each well at 100 μL, and CD16 antibody was added to each well at 15 μL. The plates were then co-cultured for 24 h.

[0079] Example 5

[0080] S1: Isolation of peripheral blood mononuclear cells:

[0081] S11. Collect peripheral blood from the healthy elbow vein, add heparin sodium for anticoagulation, centrifuge the whole blood system at room temperature at 800g for 10 minutes, then set the speed to 1 to separate cells and plasma. The upper layer is the plasma layer and the lower layer is the blood cell layer. Take the upper plasma layer, place it in a centrifuge tube, seal it, and place it in a water bath for inactivation.

[0082] S12. After inactivation, the plasma was placed at 4°C for 30 minutes and then centrifuged at 1000g for 10 minutes at room temperature (8°C for ascending and 5°C for descending). The upper plasma layer was transferred to a new centrifuge tube and stored at 4°C to obtain autologous serum. 0.9% physiological saline was injected into the lower plasma layer and mixed evenly to obtain a blood cell suspension.

[0083] S13. Take the blood cell suspension described in step S12 and inject it into 20 mL of lymphocyte separation medium. Centrifuge at 800 g for 20 min at room temperature, with a 2-degree incline and 1-degree incline. Transfer the mononuclear cell layer to a new centrifuge tube, add 0.9% physiological saline, mix well, and centrifuge at 700 g for 10 min at room temperature, with a 9-degree incline and 9-degree incline. Discard the supernatant, add 0.9% physiological saline to resuspend the cells, repeat the centrifugation steps, discard the supernatant, add RPMI-1650 medium to prepare a cell suspension, and store it at 4°C for later use.

[0084] S2: Preparation of induction culture medium:

[0085] S21. Astragalus membranaceus and Eucommia ulmoides were pulverized separately to obtain Astragalus membranaceus powder and Eucommia ulmoides powder respectively. 4g of Astragalus membranaceus powder and 1g of Eucommia ulmoides powder were mixed and added to a round-bottom flask. 100mL of purified water was added and the temperature was raised to 85℃ for water bath heating and reflux reaction. After the reaction was carried out for 2 hours, the mixture was filtered, the filtrate was collected, concentrated to 1 / 3 of the original volume, and ethanol solution was added for alcohol precipitation. After standing overnight, the mixture was filtered, the precipitate was collected, and the complex polysaccharide was obtained.

[0086] S22. After washing the beets, peel and cut them into pieces. Place them in a low-speed juicer and juice them at 50 rpm. After filtering, collect the filtrate to obtain beet juice.

[0087] S23. Activate *Lactobacillus plantarum*, spread it on MRS agar plates, pick a single colony and place it in MRS broth medium. After incubation at 37°C for 24 hours, obtain the bacterial suspension with a viable cell concentration of 1.12 × 10⁻⁶. 8 CFU / mL;

[0088] S24. Mix 10 mL of beet juice prepared in step S2 and 1.5 g of complex polysaccharide prepared in step S21. Add 15% by volume to the bacterial solution prepared in step S23. Ferment at 36°C for 24 h. Centrifuge the fermentation broth at 7000 rpm for 10 min at 4°C. Sterilize at 90°C for 1 h. Cool and store at 4°C for later use to obtain the induction culture medium.

[0089] S3: Cell Culture

[0090] S31. Human nephroblastoma cell lines were cultured in McCoy's 5A medium containing 15% FBS and 1% P / S, and then neutrally cultured in an incubator at 37°C and 5% CO2. When the cell density reached 72.8%, trypsin digestion solution containing 0.25% EDTA was added for digestion, and then passaged.

[0091] S32. Dilute the peripheral blood mononuclear cells obtained in step S1 to 1.17 × 10⁻⁶ using PRMI-1640 containing 10% FBS. 6 cells / mL, at a rate of 1.07 × 10⁻⁶ per well. 3 Peripheral blood mononuclear cells were transferred to co-culture chambers at 1 per well.

[0092] S33, human nephroblastoma cells were divided into groups of 1.83 × 10⁻⁶. 3 Seeds were seeded per well in a 6-well plate. A suspended co-culture chamber was placed in each well and incubated at 37°C and 5% CO2. Induction culture medium prepared in step S2 was added to each well at 100 μL, and CD16 antibody was added to each well at 15 μL. Co-culture was then carried out.

[0093] Experimental Example 1

[0094] The NK cells cultured in Examples 1-5 were analyzed for relevant NK cell manifestations using flow cytometry. 20 μL of the NK cell suspension from Examples 1-5 was diluted 1-fold, and 1.0 × 10⁷ NK cells were placed in a T25 culture flask and cultured at 37°C in a 5% CO₂ incubator for 4 h. Cells were then collected by centrifugation at 500g for 5 min, and 10 mL of 1×PBS was added. The cells were then resuspended in 1×PBS to prepare a single-cell suspension. Fluorescently labeled antibodies were used to stain for relevant NK cell activity molecules. The flow cytometry antibodies used included: KLRK1 Antibody (Human), Qinke Biotechnology, catalog number: DF4816; DR4 Antibody (Human), Qinke Biotechnology, catalog number: AF0304; CD69 Antibody (Human), Qinke Biotechnology, catalog number: AF5275; NCR3 Antibody (Human), Qinke Biotechnology, catalog number: DF4221; LAMP1 Antibody (Human), Qinke Biotechnology, catalog number: DF4806; CD25 Antibody (Human), Qinke Biotechnology, catalog number: AF7675;

[0095] Figure 1The diagram shows the expression levels of various molecules in NK cells cultured in Examples 1-5. KLRK1 (NKG2D) is an important activating receptor that recognizes stress molecules on tumor or infected cells. Activation of this receptor can enhance the cytotoxicity of NK cells and promote their proliferation and function. Activation of the NKG2D signaling pathway helps increase the effector functions of NK cells, such as cell killing and cytokine secretion. DR4 is a member of the tumor necrosis factor receptor superfamily and mainly activates the apoptosis pathway in NK cells by binding to its ligand (such as TRAIL). CD69 is an early activation marker molecule, which is usually upregulated after NK cells are stimulated. CD69 expression is closely related to NK cell activation, proliferation, and immune response. By promoting early activation of NK cells, CD69 helps enhance cytokine secretion and supports NK cell proliferation and activity. NCR3 (NKp30) is an activating receptor for NK cells, involved in recognizing virus-infected cells and tumor cells. Activation of NCR3 can enhance NK cell activity. The cytotoxicity and cytokine secretion of NK cells promote their proliferation and activity; LAMP1 is a component of the lysosomal membrane and is closely related to the cytotoxic function of NK cells; LAMP1 expression is related to the killing function of NK cells, which can promote the fusion of lysosomes with target cells and release cytotoxic molecules (such as perforin and Granzie enzyme), thereby enhancing the effector function of NK cells; CD25 is the α chain of the IL-2 receptor and participates in the proliferation and survival of NK cells. After IL-2 binds to CD25, it can activate the proliferation of NK cells and enhance their cytokine secretion and cytotoxic effects; as shown in the figure, Examples 3-5 can significantly improve the expression levels of NKG2D, CD69, NKp30 and CD25. Example 1 has a significant effect on improving the expression of NKG2D, but it is slightly weak for the expression of other molecules. In Example 2, the expression of NKG2D and LAMP1 is somewhat lacking. It can be seen that the complex polysaccharide of Astragalus membranaceus and Eucommia ulmoides can induce the increase in the expression level of related functional molecules. At the same time, the human nephroblastoma cells secrete IL-2. Co-culture with IL-15 can significantly increase the proliferation rate of NK cells, increase the expression of activation markers (such as CD69 and NKG2D) by NK cells, and enhance their anti-tumor effects. After co-culture, the killing rate of NK cells against tumor cells such as K562 is significantly increased.

[0096] Experimental Example 2

[0097] This experiment used ELISA to detect interferon expression, using 1×10⁶ NK cells cultured in Examples 1-5. 4IFN-γ was co-cultured with gastric cancer tumor cells at a target-efficacy ratio of 1:1 in 96-well plates under the conditions of 37°C, 5% CO2, and culture time of 48 h. The supernatant was collected after co-culture and the IFN-γ concentration was measured using a double-antibody sandwich ELISA kit (Elabscience).

[0098] Figure 2 The figure shows the results of IFN-γ release capacity of NK cells cultured in Examples 1-5. As shown in the figure, there is a significant difference in IFN-γ release capacity between Examples 3-5 and Examples 1 and 2. This is related to the upregulation of the expression of activating molecules on NK cells. However, in Experiment 1, there is no significant difference in the expression of NKD2D (KLTK1) molecules. The IFN-γ release capacity is also related to the upregulation of functional molecules of the NCRs family. Examples 3-5 have a clear advantage in the expression of NRC3 functional molecules. Therefore, the IFN-γ release capacity is also significantly improved.

[0099] Experimental Example 3

[0100] This experiment investigated the cytotoxic activity of NK cells cultured in Examples 1-5. K562 cells were used as the target cells. K562 tumor cell lines were revived at 37°C. The revived K562 target cells were then cultured in PRIM-1640 medium containing 10% FBS at a density of 1×10⁵ cells / mL, and cultured at 37°C and 5% CO₂. 20 μL of K562 target cell suspension was diluted to a cell density of 1×10⁵ cells / mL and added to 12-well plates at a target-efficacy ratio of 10:1. Single-cell K562 target cell culture was used as a control. After co-culturing at 37°C and 5% CO₂ for 4 hours, cells were collected, and the cytotoxic activity of NK cells was detected using flow cytometry. The formula for calculating the cytotoxic activity of NK cells is shown below:

[0101] NK cell activity (%) = ×100%;

[0102] Figure 3The figure shows the cytotoxic activity of K562 target cells of NK cells cultured in Examples 1-5 of this invention. As shown in the figure, the cytotoxic activity of Example 1 was the lowest at 0d, 7d and 10d. Example 2 showed a significant improvement compared to Example 1, but there was no significant difference in cytotoxic activity between 7d and 10d in Example 2. The cytotoxic activity of NK cells cultured in Examples 3-5 was relatively high. In Examples 3-5, fermentation broth containing Astragalus membranaceus and Eucommia ulmoides polysaccharides was added to the co-culture chamber. The fermentation of Lactobacillus plantarum can convert glucose and polysaccharides in beet juice into short-chain fatty acids such as lactic acid, acetic acid, propionic acid, and butyric acid. This can induce the activation of NADH-ubiquinone oxidoreductase, a key gene in the proliferation and development of NK cells, and can promote cell growth, differentiation, and energy metabolism, thereby enhancing NK cell activity. When NK cells are co-cultured with human nephroblastoma cells, they secrete IL-2 and IL-15, which can significantly increase the proliferation rate of NK cells. Co-culturing with NK cells can significantly increase the number of NK cells. Human nephroblastoma cells can increase the expression of activation markers (such as CD69 and NKG2D) of NK cells, thereby enhancing their anti-tumor effect. After co-culture, the killing rate of NK cells against tumor cells such as K562 is significantly increased.

[0103] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

[0104] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for culturing induced NK cells, characterized in that: Specifically, the following steps are included: S1: Isolation of peripheral blood mononuclear cells; peripheral blood from healthy elbow veins was collected, the upper plasma layer was aspirated, placed in a centrifuge tube, sealed, and placed in a water bath to inactivate and separate cells and plasma. After standing and centrifugation, the lower plasma layer was collected and physiological saline was added to obtain a blood cell suspension. Lymphocyte separation medium was injected into the blood cell suspension, centrifuged, and the mononuclear cell layer was collected. Physiological saline was added, and after centrifugation and washing, culture medium was added to prepare a cell suspension for later use. S11. Take peripheral blood from the antecubital vein of a healthy person, add heparin sodium for anticoagulation, centrifuge the whole blood system at room temperature at 700-800g for 10 minutes, then set the speed to 1 to separate cells and plasma. The upper layer is the plasma layer and the lower layer is the blood cell layer. Take the upper plasma layer, place it in a centrifuge tube, seal it, and place it in a water bath for inactivation. S12. After inactivation, the plasma was placed at 4℃ for 30 minutes and then centrifuged at 900-1000g for 10 minutes at room temperature with an elevation of 8 and a descent of 5. The upper plasma layer was transferred to a new centrifuge tube and stored at 4℃ to obtain autologous serum. 0.9% physiological saline was injected into the lower plasma layer and mixed evenly to obtain a blood cell suspension. S13. Take the blood cell suspension described in step S12 and inject it into 10-20 mL of lymphocyte separation medium. Centrifuge at 700-800 g for 20 min at room temperature, with a 2-fold increase and 1-fold decrease. Transfer the mononuclear cell layer to a new centrifuge tube, add 0.9% physiological saline, mix well, and centrifuge at 700-800 g for 10 min at room temperature, with a 9-fold increase and 9-fold decrease. Discard the supernatant, add 0.9% physiological saline to resuspend the cells, repeat the centrifugation steps, discard the supernatant, add RPMI-1650 medium to prepare a cell suspension, and store it at 4°C for later use. S2: Preparation of induction culture medium; extract complex polysaccharides from Astragalus membranaceus and Eucommia ulmoides, juice beet and mix with complex polysaccharides, ferment with Lactobacillus plantarum, collect the fermentation broth, centrifuge and sterilize to obtain induction culture medium for later use; S21. Astragalus membranaceus and Eucommia ulmoides were pulverized separately to obtain Astragalus membranaceus powder and Eucommia ulmoides powder respectively. The Astragalus membranaceus powder and Eucommia ulmoides powder were mixed and added to a round bottom flask. Purified water was added and the temperature was raised to 70-100℃ for water bath heating and reflux reaction. After reacting for 1-4 hours, the mixture was filtered, the filtrate was collected, concentrated to 1 / 3 of the original volume, and ethanol solution was added for alcohol precipitation. After standing overnight, the mixture was filtered, the precipitate was collected, and the complex polysaccharide was obtained. S22. After washing the beets, peel and cut them into pieces. Place them in a low-speed juicer and juice them at a speed of 50-80 rpm. After filtering, collect the filtrate to obtain beet juice. S23. Activate *Lactobacillus plantarum*, spread it on MRS agar plates, pick a single colony and place it in MRS broth medium. Incubate at 37°C for 24 hours to obtain a bacterial suspension with a viable cell concentration of 1.0 × 10⁻⁶. 8 -5.0×10 8 CFU / mL; S24. Mix the beet juice prepared in step S22 and the complex polysaccharide prepared in step S21, add the bacterial solution prepared in step S23 at a volume fraction of 1%-5%, place it at 33-38℃ and ferment for 18-36 hours. Then, centrifuge the fermentation broth at 4℃ at a speed of 6000-8000 rpm for 5 minutes, raise the temperature to 90℃ for sterilization for 0.5-1 hour, cool it, and store it at 4℃ for later use to obtain the induction culture medium. S3: Cell culture: Human nephroblastoma cells and the cell suspension prepared in step S1 were co-cultured, and the induction culture medium prepared in step S2 was added for induction. S31. Human nephroblastoma cell lines were cultured in McCoy's 5A medium containing 15% FBS and 1% P / S, and then neutrally cultured in an incubator at 37°C and 5% CO2. When the cell density reached 70%-90%, trypsin digestion solution containing 0.25% EDTA was added for digestion, and then passaged. S32. Dilute the peripheral blood mononuclear cells obtained in step S1 to 1.0 × 10⁻⁶ using PRMI-1640 containing 10% FBS. 6 cells / mL, at a rate of 1.0 × 10⁻⁶ per well. 3 -2.0×10 3 Peripheral blood mononuclear cells were transferred to co-culture chambers at 1 per well. S33, human nephroblastoma cells were divided into groups of 1.0 × 10⁻⁶. 3 -2.0×10 3 Seeds were seeded per well in a 6-well plate. A suspended co-culture chamber was placed in each well and incubated at 37°C and 5% CO2. Induction culture medium prepared in step S2 was added to each well at 100 μL, and CD16 antibody was added to each well at 15 μL. The plates were then co-cultured for 24 h.

2. The method for culturing induced NK cells according to claim 1, characterized in that: In step S21, the mass ratio of Astragalus powder to Eucommia powder is 3-5:

1.

3. The method for culturing induced NK cells according to claim 2, characterized in that: In step S24, the mass concentration of the complex polysaccharide in the beet juice is 0.05-0.15 g / mL.

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