NK cell culture method

Abstract

The invention provides a culture method of NK cells, and belongs to the field of cytobiology and immunology. The culture method of the NK cells comprises the following steps: resuspending NK cell source cells by using an induction culture medium, then inoculating the NK cell source cells into an induction culture bottle, culturing to obtain a cell culture solution after the culture is completed, then adding an amplification culture medium into the cell culture solution for culturing, and collecting the NK cells after the culture is completed, wherein the induction culture medium comprises an AIMV culture medium and a first active component, and the first active component comprises a yeast extract and NAD < + >; the amplification culture medium comprises a DMEM culture medium and a second active component, and the second active component comprises a TGF beta inhibitor, tanshinone and L-alanine. The NK cells obtained by the culture method of the NK cells have higher cell purity and cell quantity and stronger killing activity; in addition, the NK cells obtained by the culture method of the NK cells can keep activity for a long time.

Description

Technical Field

[0001] The present application relates to the fields of cell biology and immunology, and in particular to a method for culturing NK cells. Background Art

[0002] Cancer is a major health concern and has become the second leading cause of death worldwide. Cancer treatments primarily include traditional surgery, radiotherapy, chemotherapy, and newer treatments that have seen rapid growth in recent years, such as targeted therapy and immunotherapy.

[0003] Tumor immunotherapy is a treatment method that applies immunological principles and methods to activate or enhance the body's anti-tumor immune response to kill tumor cells and inhibit tumor growth. This treatment approach is fundamentally different from traditional treatments such as surgery, chemotherapy, and radiotherapy. Instead of directly killing cancer cells, it mobilizes the body's tumor-recognizing immune cells, enhancing the immune system's combat capability and indirectly killing and controlling cancer. It has minimal side effects and is safe and effective.

[0004] The main approaches to tumor immunotherapy include molecular targeted therapy, immune checkpoint inhibitors (such as PD-1 / PD-L1 and CTLA-4 inhibitors), adoptive immune cell therapy (such as CAR-T, TIL, NK, CIK / DC-CIK), cytokine therapy, and tumor vaccines. These treatments activate or enhance the body's anti-tumor immune response through different mechanisms, achieving the therapeutic effect of controlling or even specifically eliminating tumors.

[0005] NK cells are an important component of the human body's natural immune system and play an important role in the occurrence and development of tumors. They have unique anti-tumor effects, including MHC-unrestricted cytotoxicity, cytokine production, and immune memory, making them key players in the innate and adaptive immune response systems. NK cells have unique inflammatory tropism and can migrate to tumor sites in response to cytokines and chemokines released by the tumor microenvironment, demonstrating unique advantages in tumor targeted therapy. In addition, NK cell adoptive therapy or CAR-NK cell therapy rarely produces graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), and is more effective than CAR-T therapy in treating solid tumors.

[0006] The effectiveness of cell therapy is primarily influenced by factors such as cell number, cytotoxic activity, and survival rate. Current in vitro NK cell culture protocols generally suffer from low induction efficiency and insufficient cytotoxic activity, which limits therapeutic efficacy. Therefore, optimizing NK cell culture methods is crucial to improve their induction efficiency and cytotoxic activity, thereby enhancing therapeutic efficacy. Summary of the Invention

[0007] The present application provides a method for culturing NK cells, which can effectively improve the purity and viability of the obtained NK cells, reduce the inhibition of NK cells in the tumor killing process, and ultimately improve the killing activity of NK cells against tumor cells.

[0008] The embodiment of the present application is implemented as follows: In the first aspect, the present application example provides a method for culturing NK cells, which includes: first resuspending NK cell-derived cells using an induction medium, then inoculating them into an induction culture bottle for a first culture, obtaining a cell culture fluid after the first culture is completed, then adding an expansion medium to the cell culture fluid for a second culture, and collecting NK cells after the second culture is completed; wherein, the induction medium includes AIMV medium and a first active ingredient, the first active ingredient includes yeast extract and NAD+; the expansion medium includes DMEM medium and a second active ingredient, the second active ingredient includes TGFβ inhibitor, tanshinone and L-alanine.

[0009] In the above technical scheme, the NK cell culture method of the present application first resuspends the NK cell source cells with a special induction culture medium, completes the first culture, and then performs a second culture with a special expansion culture medium. The obtained NK cells have higher cell purity and cell number and stronger killing activity. On the one hand, high-purity NK cells can reduce off-target effects and enhance the killing effect on target cells. On the other hand, NK cells with stronger killing activity can more effectively identify and kill tumor cells or virus-infected cells; in addition, the NK cells obtained using the NK cell culture method of the present application can remain active for a long time.

[0010] In some possible embodiments, the concentration of yeast extract in the induction medium is 0.1-0.3 g / L, and / or the concentration of NAD+ in the induction medium is 8.0-10.0 mmol / L.

[0011] In the above technical solution, by making the concentration of yeast extract in the induction medium and / or the concentration of NAD+ in the induction medium within the above range, it is beneficial to promote the induced differentiation of NK cells.

[0012] In some possible embodiments, the induction medium further comprises 800-1000 IU / mL IL-2 and 80-100 ng / mL IL-15.

[0013] In the above technical solution, IL-2 and IL-15 can synergistically promote the proliferation of NK cells and can have a positive regulatory effect on the function of NK cells.

[0014] In some possible embodiments, the concentration of the TGFβ inhibitor in the expansion medium is 20-30 mg / mL, the concentration of tanshinone in the expansion medium is 0.8-1.0 mg / mL, and the concentration of L-alanine in the expansion medium is 5-10 μg / mL.

[0015] In the above technical solution, by making at least one of the concentrations of TGFβ inhibitor, tanshinone and L-alanine in the expansion medium within the above range, it is beneficial to promote the expansion and activation of NK cells.

[0016] In some possible embodiments, the expansion medium further comprises 200-300 IU / mL IL-12, 80-100 ng / mL IL-15, 8-12 ng / mL IL-18, and 90-120 IU / mL IL-21.

[0017] In some possible embodiments, the induction culture flask is treated with a coating solution, the coating solution includes AIMV culture medium and anti-CD3 antibody, and the concentration of the anti-CD3 antibody in the coating solution is 1-10 μg / mL.

[0018] In some possible embodiments, the volume of the induction medium for resuspending the NK cell-derived cells is 22-25 mL.

[0019] In the above technical solution, by making the volume of the induction medium for resuspending NK cell-derived cells within the above range, it is possible to ensure that the concentration of each component in the induction medium is sufficient and avoid cytotoxicity caused by excessive amounts of each component.

[0020] In some possible embodiments, the first culture and the second culture are performed in a carbon dioxide constant temperature incubator, and the environment in the carbon dioxide incubator is 37° C. and 5% CO 2 ; Optionally, the first culture period is 3 to 5 days; Optionally, the second culture period is 13 to 17 days.

[0021] In some possible embodiments, the cell density during the second culture is 1.0×10 6 ~2.0×10 6 pieces / mL.

[0022] In the above technical solution, by keeping the cell density in the above range during the second culture process, NK cells have stronger killing activity, thereby being able to more effectively identify and kill tumor cells or virus-infected cells.

[0023] In some possible embodiments, the NK cell-derived cells include at least one of peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, induced pluripotent stem cells, and embryonic stem cells.

[0024] In the above technical solution, the source of NK cell-derived cells is wide and easy to standardize, which has important clinical significance for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 This is a flow cytometry analysis result of NK cells cultured in Example 1 of the present invention; Figure 2 This is a flow cytometry analysis result of NK cells cultured in Comparative Example 1 of the present invention; Figure 3 This is a flow cytometry analysis result diagram of NK cells cultured in Comparative Example 2 of the present invention; Figure 4 This is a flow cytometry analysis result of NK cells cultured in Comparative Example 3 of the present invention; Figure 5 This is a flow cytometry analysis result of NK cells cultured in Comparative Example 4 of the present invention; Figure 6 This is a flow cytometry analysis result of NK cells cultured in Comparative Example 5 of the present invention; Figure 7 This is a flow cytometry analysis result diagram of NK cells cultured in Comparative Example 6 of the present invention. DETAILED DESCRIPTION

[0027] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer recommendations. The reagents or instruments used are not specified by the manufacturer and are conventional products that can be purchased commercially.

[0028] The following is a detailed description of a method for culturing NK cells in accordance with an embodiment of the present application: The present application provides a method for culturing NK cells, which comprises the following steps: S1. Obtaining NK cell-derived cells NK cell-derived cells include at least one of peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, induced pluripotent stem cells, and embryonic stem cells.

[0029] The method for isolating and obtaining peripheral blood mononuclear cells from peripheral blood is as follows: Under a sterile environment, 60-80 mL of human peripheral blood was obtained, diluted with PBS buffer, and subjected to differential centrifugation to obtain peripheral blood mononuclear cells.

[0030] The NK cell culture method of the present application has a wide range of sources of NK cell-derived cells, is easy to standardize, and has important clinical significance for tumor immunotherapy.

[0031] S2. First cultivation First, the NK cell-derived cells are resuspended in an induction culture medium, and then inoculated into an induction culture flask for the first culture. After the first culture is completed, a cell culture fluid is obtained.

[0032] The induction culture medium includes AIMV culture medium and a first active ingredient, and the first active ingredient includes yeast extract and NAD+.

[0033] Yeast extract has the dual functions of nutritional supplementation and immune activation in the induction culture medium. It can not only promote the proliferation of NK cells, but also enhance their anti-tumor activity through components such as β-glucan.

[0034] NAD+ has the dual functions of metabolic regulation and immune activation in the induction culture medium. It can significantly enhance its anti-tumor effect and maintain its homeostasis by improving mitochondrial function, enhancing cytotoxicity, promoting proliferation and reversing immunosuppression.

[0035] Optionally, the concentration of yeast extract in the induction medium is 0.1-0.3 g / L, and / or the concentration of NAD+ in the induction medium is 8.0-10.0 mmol / L.

[0036] As an example, the concentration of yeast extract in the induction medium can be 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L or 0.3 g / L; the concentration of NAD+ in the induction medium can be 8.0 mmol / L, 8.5 mmol / L, 9.0 mmol / L, 9.5 mmol / L or 10.0 mmol / L.

[0037] Optionally, the induction medium further comprises 800-1000 IU / mL IL-2 and 80-100 ng / mL IL-15.

[0038] IL-2 and IL-15 can synergistically promote the proliferation of NK cells and have a positive regulatory effect on the function of NK cells.

[0039] As an example, the concentration of IL-2 in the induction medium can be 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL or 1000 IU / mL; the concentration of IL-15 in the induction medium can be 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL or 100 ng / mL.

[0040] Optionally, resuspend the NK cell-derived cells in an induction medium volume of 22-25 mL.

[0041] As an example, the volume of the induction medium for resuspending the NK cell-derived cells can be 22 mL, 23 mL, 24 mL, or 25 mL.

[0042] By setting the volume of the induction medium for resuspending NK cell-derived cells within the above range, it is possible to ensure that the concentration of each component in the induction medium is sufficient, and to avoid cytotoxicity caused by excessive amounts of each component.

[0043] The induction culture flask is treated with a coating solution comprising an AIMV culture medium and an anti-CD3 antibody. The concentration of the anti-CD3 antibody in the coating solution is 1-10 μg / mL.

[0044] As an example, the concentration of the anti-CD3 antibody in the coating solution may be 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL or 10 μg / mL.

[0045] The method for treating culture flasks with coating solution to obtain induction culture flasks is as follows: Anti-CD3 antibody was added to AIMV culture medium and added to T75 cell culture flask for coating, cultured overnight at 4°C, and the coated culture flask was used as an induction culture flask.

[0046] The first culture was carried out in a carbon dioxide constant temperature incubator with an environment of 37°C and 5% CO2 for 3 to 5 days.

[0047] As an example, the first culture may be cultured for 3 days, 4 days, or 5 days.

[0048] S3, Second Cultivation An expansion medium is added to the cell culture fluid for a second culture, and the expansion medium is supplemented to the cell culture fluid according to the cell density during the culture process. After the second culture is completed, the NK cells are collected.

[0049] The expansion culture medium includes DMEM culture medium and a second active ingredient, and the second active ingredient includes a TGFβ inhibitor, tanshinone and L-alanine.

[0050] TGFβ inhibitors can further enhance the anti-tumor ability of NK cells and their persistence in the body by blocking TGF-β signaling.

[0051] Tanshinone and L-alanine can synergistically promote the expansion and activation of NK cells.

[0052] Optionally, the concentration of the TGFβ inhibitor in the expansion medium is 20-30 mg / mL, and / or the concentration of tanshinone in the expansion medium is 0.8-1.0 mg / mL, and / or the concentration of L-alanine in the expansion medium is 5-10 μg / mL.

[0053] As an example, the concentration of TGFβ inhibitor in the expansion medium can be 20 mg / mL, 22 mg / mL, 25 mg / mL, 28 mg / mL or 30 mg / mL; the concentration of tanshinone in the expansion medium can be 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL or 1.0 mg / mL; the concentration of L-alanine in the expansion medium can be 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL.

[0054] Optionally, the expansion medium further comprises 200-300 IU / mL IL-12, 80-100 ng / mL IL-15, 8-12 ng / mL IL-18, and 90-120 IU / mL IL-21.

[0055] The combined use of IL-12, IL-15, and IL-18 as active ingredients in the expansion culture medium can reduce dependence on high concentrations of IL-2, reduce the side effects of regulatory T cells (Treg), and at the same time increase the expansion multiples and killing activity of NK cells.

[0056] IL-21 plays an important role in inducing NK cell maturation and enhancing its cytotoxicity.

[0057] As an example, the concentration of IL-12 in the expansion medium can be 200 IU / mL, 220 IU / mL, 250 IU / mL, 280 IU / mL or 300 IU / mL; the concentration of IL-15 in the expansion medium can be 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL or 100 ng / mL; the concentration of IL-18 in the expansion medium can be 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL or 12 ng / mL; and the concentration of IL-21 in the expansion medium can be 90 IU / mL, 100 IU / mL, 110 IU / mL or 120 IU / mL.

[0058] The second culture was carried out in a carbon dioxide constant temperature incubator with an environment of 37°C and 5% CO2 for 13 to 17 days.

[0059] As an example, the second culture may be carried out for 15 days.

[0060] Optionally, the cell density during the second culture was 1.0 × 10 6 ~2.0×10 6 pieces / mL.

[0061] By keeping the cell density within the above range during the second culture process, NK cells have stronger killing activity, thereby being able to more effectively identify and kill tumor cells or virus-infected cells.

[0062] The NK cell culture method of the present application first resuspends the NK cell source cells in a special induction culture medium, completes the first culture, and then performs a second culture in a special expansion culture medium. The obtained NK cells have higher cell purity and cell number and stronger killing activity. On the one hand, high-purity NK cells can reduce off-target effects and enhance the killing effect on target cells. On the other hand, IL-2 and IL-5 added during the culture process can effectively activate NK cells and enhance the ADCC effect mediated by them, so that they can more effectively identify and kill tumor cells or virus-infected cells in immunotherapy; in addition, the NK cells obtained using the NK cell culture method of the present application can remain active for a long time.

[0063] The following is a further detailed description of a NK cell culture method of the present application in conjunction with the examples.

[0064] Example 1 The present invention provides a method for culturing NK cells, which comprises the following steps: S1. Obtain peripheral blood mononuclear cells 60 mL of peripheral blood was collected from subjects without autoimmune diseases, tumors, viral or bacterial infections. Heparin anticoagulation was accelerated and the blood was centrifuged at 800 × g for 10 min. The upper plasma layer was aspirated and set aside. PBS buffer was added to the lower cells to make the volume 60 mL. 30 mL of Ficoll separation solution was added to a sterile centrifuge tube. The above-mentioned fixed volume liquid was then slowly added to the top of the Ficoll separation solution. The tube was centrifuged at 700 × g for 20 min. The upper plasma layer was carefully removed, and the middle buffy coat cells were aspirated. The cells were then washed twice with 3 times PBS buffer and centrifuged at 600 × g for 10 min. The resulting precipitate was the peripheral blood mononuclear cells.

[0065] S2. First cultivation The peripheral blood mononuclear cells obtained in step S1 were resuspended in induction medium and then cultured at a rate of 1.5×10 6 The cells were inoculated at a density of 100 cells / mL into the coated induction culture flask and induced for 3 days in a 37°C, 5% CO2 incubator to obtain cell culture fluid.

[0066] The induction medium included AIMV medium, 0.2 g / L yeast extract, 9 mmol / L NAD+, 900 IU / mL IL-2, and 90 ng / mL IL-15.

[0067] The induction culture medium consists of AIMV culture medium and anti-CD3 antibodies added thereto. The concentration of the anti-CD3 antibodies in the coating solution is 2.5 μg / mL. The method for treating the culture flask with the coating solution to obtain the induction culture flask is as follows: add anti-CD3 antibodies to the AIMV culture medium and add it to the T75 cell culture flask for coating, culture overnight at 4°C, and use the coated culture flask as the induction culture flask.

[0068] S3, Second Cultivation Add expansion medium to the cell culture medium obtained in step S2 and continue culturing in a 37°C, 5% CO2 incubator for 12 days. Regularly replenish expansion medium to ensure that the cell density is maintained at 1.0×10 6 ~2.0×10 6 / mL, and NK cells were obtained.

[0069] The expansion medium included DMEM medium, 25 mg / mL TGFβ inhibitor, 9 mg / mL tanshinone, 5 μg / mL L-alanine, 250 IU / mL IL-12, 90 ng / mL IL-15, 10 ng / mL IL-18, and 110 IU / mL IL-21.

[0070] Example 2 The present embodiment provides a method for culturing NK cells, which changes the formula of the induction medium based on Example 1, which includes AIMV medium, 0.1 g / L yeast extract, 8 mmol / L NAD+, 800 IU / mL IL-2 and 80 ng / mL IL-15, while the other ingredients remain unchanged.

[0071] Example 3 The present embodiment provides a method for culturing NK cells, which changes the formula of the induction medium based on Example 1, which includes AIMV medium, 0.3 g / L yeast extract, 10 mmol / L NAD+, 1000 IU / mL IL-2 and 100 ng / mL IL-15, while the other ingredients remain unchanged.

[0072] Example 4 The present application provides a method for culturing NK cells, which changes the formula of the expansion medium based on Example 1, comprising DMEM medium, 20 mg / mL TGFβ inhibitor, 0.8 mg / mL tanshinone, 5 μg / mL L-alanine, 200 IU / mL IL-12, 80 ng / mL IL-15, 8 ng / mL IL-18 and 90 IU / mL IL-21, while the others remain unchanged.

[0073] Example 5 The present application provides a method for culturing NK cells, which changes the formula of the expansion medium based on Example 1, comprising DMEM medium, 30 mg / mL TGFβ inhibitor, 1.0 mg / mL tanshinone, 10 μg / mL L-alanine, 300 IU / mL IL-12, 100 ng / mL IL-15, 12 ng / mL IL-18 and 120 IU / mL IL-21, while the others remain unchanged.

[0074] Example 6 An embodiment of the present application provides a method for culturing NK cells, which changes the formula of the induction medium based on Example 1, wherein IL-2 and IL-15 are not added, that is, the induction medium includes AIMV medium, 0.2 g / L yeast extract and 9 mmol / L NAD+, and the other factors remain unchanged.

[0075] Example 7 An embodiment of the present application provides a method for culturing NK cells, which changes the formula of the expansion medium based on Example 1, wherein IL-12, IL-15, IL-18 and IL-21 are not added, that is, the expansion medium includes DMEM medium, 25 mg / mL TGFβ inhibitor, 9 mg / mL tanshinone and 5 μg / mL L-alanine, and the rest remains unchanged.

[0076] Comparative Example 1 The comparative example of the present application provides a method for culturing NK cells, which changes the formula of the induction medium based on Example 1, wherein no yeast extract is added, that is, the induction medium includes AIMV medium, 9 mmol / L NAD+, 900 IU / mL IL-2 and 90 ng / mL IL-15.

[0077] Comparative Example 2 The comparative example of the present application provides a method for culturing NK cells, which changes the formula of the induction medium based on Example 1, wherein NAD+ is not added, that is, the induction medium includes AIMV medium, 0.2 g / L yeast extract, 900 IU / mL IL-2 and 90 ng / mL IL-15.

[0078] Comparative Example 3 The comparative example of the present application provides a method for culturing NK cells, which changes the formula of the expansion medium based on Example 1, wherein no TGFβ inhibitor is added, that is, the expansion medium includes DMEM medium, 9 mg / mL tanshinone, 5 μg / mL L-alanine, 250 IU / mL IL-12, 90 ng / mL IL-15, 10 ng / mL IL-18 and 110 IU / mL IL-21.

[0079] Comparative Example 4 The comparative example of the present application provides a method for culturing NK cells, which changes the formula of the expansion medium based on Example 1, wherein tanshinone is not added, that is, the expansion medium includes DMEM medium, 25 mg / mL TGFβ inhibitor, 5 μg / mL L-alanine, 250 IU / mL IL-12, 90 ng / mL IL-15, 10 ng / mL IL-18 and 110 IU / mL IL-21.

[0080] Comparative Example 5 The comparative example of the present application provides a method for culturing NK cells, which changes the formula of the expansion medium based on Example 1, wherein L-alanine is not added, that is, the expansion medium includes DMEM medium, 25 mg / mL TGFβ inhibitor, 9 mg / mL tanshinone, 250 IU / mL IL-12, 90 ng / mL IL-15, 10 ng / mL IL-18 and 110 IU / mL IL-21.

[0081] Comparative Example 6 The comparative example of the present application provides a method for culturing NK cells, which simultaneously changes the formula of the induction medium and the expansion medium on the basis of Example 1, wherein the induction medium includes AIMV medium, 25 mg / mL TGFβ inhibitor, 9 mg / mL tanshinone, 5 μg / mL L-alanine, 900 IU / mL IL-2 and 90 ng / mL IL-15, and the expansion medium includes DMEM medium, 0.2 g / L yeast extract, 9 mmol / L NAD+, 250 IU / mL IL-12, 90 ng / mL IL-15, 10 ng / mL IL-18 and 110 IU / mL IL-21.

[0082] Test Example 1 The NK cells obtained in Example 1 and Comparative Examples 1 to 6 were centrifuged at 500×g for 5 min, the supernatant was discarded, and then washed three times with buffer, and the cells were resuspended and adjusted to a concentration of 1.0×10 6 10 μL of phycoerythrin conjugate-labeled CD56 monoclonal antibody and 10 μL of fluorescein isothiocyanate-labeled CD3 monoclonal antibody were added to the above-mentioned cell sample (100 μL), incubated at room temperature for 15 min, and washed 3 times with PBS to remove excess antibodies. Finally, the cells were resuspended with PBS and loaded onto a flow cytometer to analyze CD3 - CD56 + The expression of Figures 1 to 7 shown.

[0083] Depend on Figures 1 to 7 The experimental results show that the CD3 - CD56 + The expression rate of cells was better than that of comparative examples 1 to 5. In comparative examples 1 to 5, yeast extract, NAD+, TGF-β inhibitor, tanshinone and L-alanine were omitted respectively. The results showed that the expression rate of CD3 - CD56 +The expression rates of the cells were lower than those in Example 1, indicating that the addition of yeast extract, NAD+, TGF-β inhibitor, tanshinone, and L-alanine would affect the expression of NK cells. In Comparative Example 6, the timing of adding yeast extract, NAD+ in the induction medium and TGF-β inhibitor, tanshinone, and L-alanine in the expansion medium was changed. The experimental results showed that the CD3 - CD56 + The expression rate of the cells is lower than that in Example 1 and Comparative Examples 1 to 6, which indicates that the timing of adding yeast extract, NAD+, TGF-β inhibitor, tanshinone, and L-alanine will also affect the expression of NK cells.

[0084] Test Example 2 CCK-8 assay to measure the killing ability of NK cells against three types of cancer organoid cells: The NK cells obtained in Example 1 and Comparative Examples 1 to 6 were centrifuged at 500×g for 5 min, the supernatant was discarded, and then washed three times with buffer, and the cells were resuspended and adjusted to a concentration of 4.0×10 6 / mL, 2.0×10 6 / mL, 1.0×10 6 / mL, 0.5×10 6 The colorectal cancer organoid cells were diluted to 1.0×10 5 The cells were then added to a 96-well plate (100 μL per well, 15 wells total) and incubated at 37°C, 5% CO2 for 1 hour. The 96-well plate containing the colorectal cancer organoids was removed, the culture medium in each well was aspirated, and the cell samples to be tested were added to the 96-well plate. Each concentration was replicated in triplicate, with 100 μL per well. For the blank and control groups, 100 μL of ull640 culture medium was added to each well and incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and washed with 1640 culture medium until the NK cells were completely removed. 100 μL of 1640 culture medium and 10 μL of CCK-8 were added to each well and incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and the OD value of each well was read at dual wavelengths of 450 nm and 630 nm.

[0085] The experimental steps for measuring the killing ability of NK cells against lung cancer organoid cells and renal cancer organoid cells using the CCK-8 method are the same as above. The results are shown in Table 1.

[0086] Killing rate (%) = [1-(target group-blank group) / (control group-blank group)] × 100%.

[0087] Table 1 Killing rate of NK cells obtained from Example 1 and Comparative Examples 1 to 6 against three types of cancer organoid cells

[0088] By analyzing the data in Table 1, it can be seen that the NK cells prepared in Example 1 have the highest killing rate against the three types of cancer organoid cells, with killing rates of 78.98%, 64.70% and 57.88% against intestinal cancer, lung cancer and kidney cancer organoid cells, respectively, which are better than the killing rates of the NK cells prepared in Comparative Examples 1 to 6 against the three types of cancer organoid cells. Among them, the NK cells obtained in Comparative Example 6 have the lowest killing rate against the three types of cancer organoid cells. It can be seen that yeast extract and NAD+ promote the induction efficiency of NK cells in the induction medium, and TGF-β inhibitor, tanshinone and L-alanine promote the amplification efficiency of NK cells in the amplification medium. The two interact with each other and significantly enhance the killing ability of NK cells against the three types of cancer organoid cells.

[0089] The foregoing description is merely a specific embodiment of the present application and is not intended to limit the present application. Persons skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for culturing NK cells, characterized in that: The NK cell culture method comprises: firstly resuspending NK cell-derived cells in an induction medium, then inoculating the NK cell-derived cells into an induction culture flask for a first culture, obtaining a cell culture fluid after the first culture, then adding an expansion medium to the cell culture fluid for a second culture, and collecting the NK cells after the second culture; Wherein, the induction medium comprises AIMV medium and a first active ingredient, and the first active ingredient comprises yeast extract and NAD+; The expansion culture medium comprises a DMEM culture medium and a second active ingredient, wherein the second active ingredient comprises a TGFβ inhibitor, tanshinone and L-alanine.

2. The method for culturing NK cells according to claim 1, wherein The concentration of the yeast extract in the induction medium is 0.1-0.3 g / L, and / or the concentration of NAD+ in the induction medium is 8.0-10.0 mmol / L.

3. The method for culturing NK cells according to claim 1, wherein The induction medium also includes 800-1000 IU / mL IL-2 and 80-100 ng / mL IL-15.

4. The method for culturing NK cells according to claim 1, wherein The concentration of the TGFβ inhibitor in the expansion medium is 20-30 mg / mL, the concentration of the tanshinone in the expansion medium is 0.8-1.0 mg / mL, and the concentration of the L-alanine in the expansion medium is 5.0-10.0 μg / mL.

5. The method for culturing NK cells according to claim 1, wherein The expansion medium also includes 200-300 IU / mL IL-12, 80-100 ng / mL IL-15, 8-12 ng / mL IL-18, and 90-120 IU / mL IL-21.

6. The method for culturing NK cells according to claim 1, wherein The induction culture flask is treated with a coating solution, wherein the coating solution comprises an AIMV culture medium and an anti-CD3 antibody, and the concentration of the anti-CD3 antibody in the coating solution is 1-10 μg / mL.

7. The method for culturing NK cells according to claim 1, wherein The volume of the induction medium for resuspending the NK cell-derived cells is 22-25 mL.

8. The method for culturing NK cells according to claim 1, wherein The first culture and the second culture are carried out in a carbon dioxide constant temperature incubator, and the environment in the carbon dioxide incubator is 37° C. and 5% CO 2 ; Optionally, the first culture is performed for 3 to 5 days; Optionally, the second culture is performed for 13 to 17 days.

9. The method for culturing NK cells according to claim 1, wherein The cell density during the second culture was 1.0×10 6 ~2.0×10 6 pieces / mL.

10. The method for culturing NK cells according to claim 1, wherein The NK cell-derived cells include at least one of peripheral blood mononuclear cells, umbilical cord blood mononuclear cells, induced pluripotent stem cells and embryonic stem cells.

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