An nk culture medium containing hypotaurine and its application in improving proliferation and killing activity of nk cells

Abstract

This invention relates to the field of NK cell culture technology, and discloses an NK culture medium containing taurine and its application in improving NK cell proliferation and killing activity. The medium includes a basal NK cell culture medium and taurine; the basal NK cell culture medium does not contain taurine and includes amino acids, vitamins, inorganic salts, trace elements, D-glucose, and sodium pyruvate. When the basal NK cell culture medium is RPMI_1640 medium, the concentration of taurine is 0.1~1mM; when the basal NK cell culture medium is IMDM medium, the concentration of taurine is 0.4~4mM. This method obtains an NK culture medium by adding taurine to the basal NK cell culture medium. This NK culture medium not only increases the number of NK cells during in vitro culture, but also significantly enhances the killing activity of the cultured NK cells against tumor cells.

Description

Technical Field

[0001] This invention relates to the field of NK cell culture technology, specifically to an NK culture medium containing taurine and its application in improving NK cell proliferation and killing activity. Background Technology

[0002] NK cells, or natural killer cells, are key innate immune cells in the human body. They can directly kill abnormal cells such as tumor cells and virus-infected cells without antigen sensitization, exhibiting a broad anti-tumor spectrum. They play a central role in anti-tumor and anti-infective immunity and are widely used in adoptive cell immunotherapy. Human NK cells mainly originate from the bone marrow, but can also differentiate and develop in secondary lymphoid organs and peripheral tissues. However, the proportion of NK cells in the peripheral blood of normal individuals is only 5%-15%, which is insufficient to meet the needs of large-scale clinical treatment. Therefore, efficiently expanding NK cells in vitro and enhancing their killing activity is crucial for promoting the clinical translation of related therapeutic technologies.

[0003] To address the aforementioned issues, existing technologies have developed various NK cell culture methods and culture medium systems: one is to add exogenous stimulating factors (such as IL-2, IL-15, CD3 monoclonal antibodies, etc.) to induce cell proliferation and activation; the other is to use RPMI1640, DMEM, and other basic culture media as a base, and add serum or serum substitutes and the above-mentioned stimulating factors to construct a composite system; the third is to adopt co-culture strategies (such as co-culturing with irradiated K562 feeder cells) to enhance the stimulation signal and improve the culture effect.

[0004] Current technologies still have significant drawbacks: First, the high cost of exogenous stimulants leads to high culture costs; second, methods such as feeder co-culture are complex to operate, have large batch-to-batch variations, and pose a risk of heterogeneous contamination; third, the cultured NK cells have limited proliferation efficiency and unstable cytotoxic activity, easily leading to functional exhaustion; fourth, serum components may introduce pathogens and complicate the culture medium composition, affecting culture consistency. Therefore, developing a culture medium with clearly defined components, controllable costs, and the ability to efficiently enhance NK cell proliferation and cytotoxic activity is an urgent problem to be solved in this field. Summary of the Invention

[0005] The present invention aims to provide an NK culture medium containing taurine and its application in improving the proliferation and killing activity of NK cells, so as to solve the technical problem that the existing optimized culture medium has limited effect on improving the amplification and killing effect of NK cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: an NK culture medium containing taurine, comprising a basic NK cell culture medium and taurine; the basic NK cell culture medium comprises amino acids, vitamins, inorganic salts, trace elements, D-glucose and sodium pyruvate.

[0007] Preferably, as an improvement, the basic NK cell culture medium is free of taurine and includes, but is not limited to, one or more combinations of: RPMI_1640 medium, IMDM medium, DMEM medium, MEMα medium, DMEM / F12 medium, Gibco_CTS_NK-Xpander medium, FUJIFILM_PRIME-XV_NK Cell CDM medium, Miltenyi Biotec_NKMACS medium, R&D Systems_ExCellerate_Human NK Cell Expansion medium, STEMCELLImmunoCult NK Cell Base medium, CellGenix_NK Cell medium, Lonza_X-VIVO 10 medium, Lonza_X-VIVO 15 medium, Corning_KBM551, Corning_KBM581, Ekosei_NK serum-free medium, Youkang_NK cell serum-free medium, Dakowei_L500 lymphocyte serum-free medium, and Dakowei_N500 NK cell serum-free medium.

[0008] Preferably, as an improvement, when the basic NK cell culture medium is RPMI-1640 medium, the concentration of the taurine is 0.1~1mM.

[0009] Preferably, as an improvement, when the basic NK cell culture medium is RPMI-1640 medium, the concentration of the taurine is 0.8~1mM.

[0010] Preferably, as an improvement, when the basic NK cell culture medium is IMDM medium, the concentration of the taurine is 0.4~4mM.

[0011] Preferably, as an improvement, when the basic NK cell culture medium is IMDM medium, the concentration of the taurine is 0.4~2mM.

[0012] Preferably, as an improvement, the RPMI_1640 culture medium comprises the following raw materials in parts by weight: 10 parts glycine; 8.9 parts L-alanine; 200 parts L-arginine; 50 parts L-asparagine; 20 parts L-aspartic acid; 65 parts L-cysteine ​​dihydrochloride; 20 parts L-glutamic acid; 15 parts L-histidine; 20 parts L-hydroxyproline; 50 parts L-isoleucine; 50 parts L-leucine; 40 parts L-lysine hydrochloride; 15 parts L-methionine; 15 parts L-phenylalanine; 20 parts L-proline; 30 parts L-serine; 20 parts L-threonine; 5 parts L-tryptophan; 2 9 parts L-tyrosine disodium salt; 20 parts L-valine; 2.5 parts ascorbate phosphate; 0.2 parts biotin; 3 parts choline chloride; 0.25 parts D-calcium pantothenate; 1 part folic acid; 1 part nicotinamide; 1 part para-aminobenzoic acid; 1 part pyridoxal hydrochloride; 0.2 parts riboflavin; 1 part thiamine hydrochloride; 0.005 parts vitamin B12; 35 parts inositol; 100 parts calcium nitrate tetrahydrate; 48.84 parts anhydrous magnesium sulfate; 400 parts potassium chloride; 2000 parts sodium bicarbonate; 6000 parts sodium chloride; 800 parts anhydrous disodium hydrogen phosphate; 0.874 parts zinc sulfate heptahydrate; 400 parts AlbuMAX® II. Serum-free culture medium supplement; 7.5 parts human transferrin; 10 parts recombinant insulin whole chain; 0.0003 parts ammonium vanadate; 0.00125 parts copper sulfate; 0.00005 parts manganese chloride; 0.005 parts sodium selenite; 2000 parts D-glucose; 1.9 parts ethanolamine; 1 part reduced glutathione; 110 parts sodium pyruvate.

[0013] Preferably, as an improvement, the IMDM culture medium comprises the following raw materials in parts by weight: 40 parts glycine; 33 parts L-alanine; 1083 parts L-alanyl-L-glutamine; 112 parts L-arginine hydrochloride; 38 parts L-asparagine; 40 parts L-aspartic acid; 93 parts L-cysteine; 100 parts L-glutamic acid; 56 parts L-histidine hydrochloride monohydrate; 140 parts L-isoleucine; 140 parts L-leucine; 195 parts L-lysine hydrochloride; 40 parts L-methionine; 88 parts L-phenylalanine; 53 parts L-proline; 56 parts L-serine; 127 parts L-threonine; 21 parts L-tryptophan; 139 parts L-tyrosine diethylcarboxylate. Sodium salt; 125 parts L-valine; 0.017 parts biotin; 5.3 parts choline chloride; 5.3 parts D-calcium pantothenate; 5.3 parts folic acid; 5.3 parts nicotinamide; 5.3 parts pyridoxal hydrochloride; 0.53 parts riboflavin; 5.3 parts thiamine hydrochloride; 0.017 parts vitamin B12; 9.6 parts inositol; 292 parts calcium chloride dihydrate; 267 parts magnesium sulfate heptahydrate; 440 parts potassium chloride; 0.10 parts potassium nitrate; 4032 parts sodium bicarbonate; 6000 parts sodium chloride; 188 parts sodium dihydrogen phosphate dihydrate; 0.023 parts sodium selenite pentahydrate; 6000 parts D-glucose; 7944 parts 4-hydroxyethylpiperazine ethanesulfonic acid; 147 parts sodium pyruvate.

[0014] Preferably, as an improvement, this solution also provides the application of NK culture medium containing taurine in enhancing NK cell proliferation and killing activity, wherein NK cells are cultured using the aforementioned NK culture medium.

[0015] The principles and advantages of this scheme are:

[0016] Compared to NK cell culture media obtained by directly adding taurine to basal NK cell culture medium, which has a relatively small effect on enhancing NK cell proliferation and killing ability, this method, by adding taurine to basal NK cell culture medium, not only increases the number of NK cells during in vitro culture but also significantly enhances the killing activity of cultured NK cells against tumor cells. The inventors analyzed that the reason is that taurine is not only a direct precursor to taurine synthesis, providing more efficient and targeted metabolic support for NK cells, but it also possesses unique antioxidant activity. This forms a "dual antioxidant system" with taurine produced intracellularly, synergistically reducing cellular stress, maintaining mitochondrial function, and potentially directly regulating key signaling pathways related to proliferation and killing. This multi-synergistic mechanism ultimately achieves simultaneous optimization and enhancement of NK cell quantity and killing function. Attached Figure Description

[0017] Figure 1 This application provides cell count data of PBMC-14 at different time points for Example 1.

[0018] Figure 2 This application provides the changes in the proportion of NK cells in PBMC-14 at different time points for Example 1.

[0019] Figure 3 Flow cytometry results on day 7 are provided for Example 1 of this application, showing the control group and the drug group supplemented with 0.8 mM taurine.

[0020] Figure 4 Flow cytometry results on day 13 for the control group and the drug group supplemented with 1.0 mM taurine in Example 1 of this application.

[0021] Figure 5 This application provides the cell proportion, amplification, and killing effect of PBMC-14 in drug groups obtained by adding different concentrations (0~1.0mM) of taurine on day 14, as shown in Example 1 of this application.

[0022] Figure 6 This application provides a comprehensive comparison score of PBMC-14 obtained from different concentrations (0~1.0mM) of taurine in Example 1 of this application.

[0023] Figure 7 This application provides cell count data of PBMC-16 at different time points for Example 2.

[0024] Figure 8 This application provides Example 2 of the changes in the proportion of NK cells of PBMC-16 at different time points.

[0025] Figure 9 Flow cytometry results on day 7 are provided for Example 2 of this application for the control group and the drug group with added 0.4 mM taurine.

[0026] Figure 10 Flow cytometry results on day 13 for Example 2 of this application, showing the control group and the drug group supplemented with 2.0 mM taurine.

[0027] Figure 11 Example 2 of this application provides the cell proportion, expansion, and killing effect of PBMC-16 with different concentrations (0~4.0mM) of taurine added on day 14.

[0028] Figure 12 Example 2 of this application provides a comprehensive comparison score of PBMC-16 in drug groups with different concentrations (0~4.0mM) of taurine.

[0029] Figure 13 This application provides cell count data for PBMC-18 at different time points for Example 3.

[0030] Figure 14 This application provides Example 3 of the changes in the proportion of NK cells of PBMC-18 at different time points.

[0031] Figure 15 Flow cytometry results on day 7 are provided for Example 3 of this application, showing the control group and the drug group supplemented with 4.0 mM taurine.

[0032] Figure 16 Flow cytometry results on day 13 are provided for Example 3 of this application, showing the control group and the drug group supplemented with 4.0 mM taurine.

[0033] Figure 17 Example 3 of this application provides the cell proportion, expansion and killing effect of PBMC-18 in drug groups obtained by adding different concentrations (0~4.0mM) of taurine on day 14.

[0034] Figure 18 Example 3 of this application provides a comprehensive comparison score of the drug groups obtained by PBMC-18 with different concentrations (0~4.0mM) of taurine.

[0035] Figure 19 Cell counts in drug groups with different concentrations (0~200μM) of taurine added are provided for Comparative Example 1 of this application.

[0036] Figure 20 This provides Comparative Example 1 of this application, showing the changes in the proportion of NK cells in drug groups obtained by adding different concentrations (0~200μM) of taurine.

[0037] Figure 21 The comprehensive comparative scores of the drug groups obtained by adding different concentrations (0~200μM) of taurine are provided for Comparative Example 1 of this application. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available.

[0039] Overview of the Plan

[0040] This solution provides an NK culture medium containing taurine, comprising a basic NK cell culture medium and taurine, wherein the basic NK cell culture medium includes amino acids, vitamins, inorganic salts, trace elements, D-glucose, and sodium pyruvate. The basic NK cell culture medium in this protocol does not contain taurine and includes, but is not limited to, one or more combinations of the following: RPMI_1640 medium, IMDM medium, DMEM medium, MEMα medium, DMEM / F12 medium, Gibco_CTS_NK-Xpander medium, FUJIFILM_PRIME-XV_NK Cell CDM medium, Miltenyi Biotec_NK MACS medium, R&D Systems_ExCellerate_Human NK Cell Expansion medium, STEMCELL ImmunoCult NK Cell Base medium, CellGenix_NK Cell medium, Lonza_X-VIVO 10 medium, Lonza_X-VIVO 15 medium, Corning_KBM551, Corning_KBM581, Ekosei_NK serum-free medium, Youkang_NK cell serum-free medium, Dakowei_L500 lymphocyte serum-free medium, and Dakowei_N500 NK cell serum-free medium.

[0041] As a reference, when the basic NK cell culture medium is RPMI_1640 medium, the concentration of taurine in the NK medium is 0.1~1mM, preferably 0.8~1mM; when the basic NK cell culture medium is IMDM medium, the concentration of taurine in the NK medium is 0.4~4mM, preferably 0.4~2mM.

[0042] For reference, RPMI_1640 medium comprises the following components by weight: 10 parts glycine; 8.9 parts L-alanine; 200 parts L-arginine; 50 parts L-asparagine; 20 parts L-aspartic acid; 65 parts L-cysteine ​​dihydrochloride; 20 parts L-glutamic acid; 15 parts L-histidine; 20 parts L-hydroxyproline; 50 parts L-isoleucine; 50 parts L-leucine; 40 parts L-lysine hydrochloride; 15 parts L-methionine; 15 parts L-phenylalanine; 20 parts L-proline; 30 parts L-serine; 20 parts L-threonine; 5 parts L-tryptophan; 29 parts L-tyrosine disodium salt; 20 parts L-valine; 2.5 parts ascorbate phosphate; 0.2 parts biotin; 3 parts choline chloride; 0.25 parts D-calcium pantothenate. ; 1 part folic acid; 1 part nicotinamide; 1 part para-aminobenzoic acid; 1 part pyridoxal hydrochloride; 0.2 parts riboflavin; 1 part thiamine hydrochloride; 0.005 parts vitamin B12; 35 parts inositol; 100 parts calcium nitrate tetrahydrate; 48.84 parts anhydrous magnesium sulfate; 400 parts potassium chloride; 2000 parts sodium bicarbonate; 6000 parts sodium chloride; 800 parts anhydrous disodium hydrogen phosphate; 0.874 parts zinc sulfate heptahydrate; 400 parts AlbuMAX® II serum-free culture medium supplement; 7.5 parts human transferrin; 10 parts recombinant insulin whole chain; 0.0003 parts ammonium vanadate; 0.00125 parts copper sulfate; 0.00005 parts manganese chloride; 0.005 parts sodium selenite; 2000 parts D-glucose; 1.9 parts ethanolamine; 1 part reduced glutathione; 110 parts sodium pyruvate.

[0043] For reference, the IMDM medium comprises the following components by weight: 40 parts glycine; 33 parts L-alanine; 1083 parts L-alanyl-L-glutamine; 112 parts L-arginine hydrochloride; 38 parts L-asparagine; 40 parts L-aspartic acid; 93 parts L-cysteine; 100 parts L-glutamic acid; 56 parts L-histidine hydrochloride monohydrate; 140 parts L-isoleucine; 140 parts L-leucine; 195 parts L-lysine hydrochloride; 40 parts L-methionine; 88 parts L-phenylalanine; 53 parts L-proline; 56 parts L-serine; 127 parts L-threonine; 21 parts L-tryptophan; 139 parts L-tyrosine. Disodium glutamate; 125 parts L-valine; 0.017 parts biotin; 5.3 parts choline chloride; 5.3 parts D-calcium pantothenate; 5.3 parts folic acid; 5.3 parts nicotinamide; 5.3 parts pyridoxal hydrochloride; 0.53 parts riboflavin; 5.3 parts thiamine hydrochloride; 0.017 parts vitamin B12; 9.6 parts inositol; 292 parts calcium chloride dihydrate; 267 parts magnesium sulfate heptahydrate; 440 parts potassium chloride; 0.10 parts potassium nitrate; 4032 parts sodium bicarbonate; 6000 parts sodium chloride; 188 parts sodium dihydrogen phosphate dihydrate; 0.023 parts sodium selenite pentahydrate; 6000 parts D-glucose; 7944 parts 4-hydroxyethylpiperazine ethanesulfonic acid; 147 parts sodium pyruvate.

[0044] This solution also provides an application of NK culture medium containing taurine in improving the proliferation and killing activity of NK cells, wherein NK cells are cultured using the above-mentioned NK culture medium.

[0045] Example 1:

[0046] I. Experimental Materials

[0047] The basic NK cell culture medium was purchased from Thermo Fisher Scientific, specifically RPMI 1640 medium, catalog number 11875093, in 500 mL form.

[0048] All reagents and kits are commercially available.

[0049] Hypotaurine was purchased from MCE, catalog number HY-100803, specification 50mg. The purchased reagent was dissolved in 458μL of sterile water to obtain a 1mol / L stock solution for later use.

[0050] II. Experimental Procedures and Results

[0051] 1. Mononuclear cell isolation and flow cytometry detection

[0052] Peripheral blood was collected from healthy volunteers. After rinsing the blood collection bag containing the sample with 75% alcohol, the blood was transferred to a 50 mL centrifuge tube. The tube was centrifuged at 800×g at room temperature for 15 min. The lower layer was then removed and diluted with an equal volume of DPBS (Durbeco phosphate-buffered saline). This diluted solution was then slowly added along the wall of the centrifuge tube at a rate of 1–3 mL / min to the upper layer of the pre-added lymphocyte separation medium, ensuring a clear interface between the blood sample and the separation medium. The mixture was then centrifuged at 600×g at room temperature for 30 min, and the white cell layer (mononuclear cells) was collected. After washing twice with DPBS, cell counting was performed at a concentration of 2.0 × 10⁻⁶ cells / mL. 6 Cells were cryopreserved. The obtained cells were then analyzed by flow cytometry to obtain NK cells (CD3+). - CD56 + / CD3 - CD16 + )Proportion.

[0053] 2. Single-factor experiment to determine the effects of taurine on NK cell proliferation and killing.

[0054] This experiment used PBMC-14 (the 14th sample of peripheral blood mononuclear cells) for comparison. Cells cultured without the drug were set as the control group. Each group had three replicates. Taurine was added to the basal NK cell culture medium at concentrations of 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, and 1.0 mM to obtain the NK cell culture medium for the drug group. The initial density of PBMC-14 was 0.5 million cells (1 × 10⁻⁶). 6 The cells were cultured in a 37°C, 5% CO2 incubator, starting with 0.5 mL of culture medium. On day 3, 0.5 mL of culture medium was added, and on day 5, 1 mL of culture medium was added. On day 7, cell counting, flow cytometry analysis, and medium replacement were performed.

[0055] Cell counting: Remove the cells from the incubator, mix well, add 10 μL of cells to 10 μL of trypan blue for staining, and count using a cell counter. Detailed cell count and NK cell percentage changes are shown below. Figure 1 As shown, the drug group supplemented with 1.0 mM taurine had better cell counts than the control group at both time points, but on day 14, the drug group supplemented with 0.8 mM taurine had the best cell counts. Figure 2 As shown, the drug groups with added 0.8mM and 1.0mM taurine concentrations had better NK cell percentages than the control group at both time points.

[0056] Flow cytometry: Take 60 μL of the mixed cells, centrifuge at 300×g for 5 min at room temperature, add 25 μL of the prepared flow cytometry antibody (PBS buffer + CD56 / CD3 / CD45 / CD16), incubate at 4℃ in the dark for 30 min, then wash with PBS buffer, centrifuge at 300×g for 5 min at room temperature, discard the supernatant, then add 200 μL of PBS buffer, mix well, incubate at room temperature in the dark for 10 min, and then detect NK cell expansion by flow cytometry. CD3 in mononuclear cells - CD56 + The proportion of NK cells in the phenotype was low, and after induction culture in NK medium containing taurine, CD3... - CD56 + The proportion of NK cells in the phenotype is significantly increased. Flow cytometry results show that, for example... Figure 3 As shown, compared with the control group on day 7, CD3 - CD56 + The phenotypic percentage increased to 29.60% in the drug group (0.8 mM taurine concentration) compared to 25.36% in the control group. Figure 4 As shown, compared with the control group on day 13, CD3 - CD56 + The phenotype rate increased to 56.64% in the drug group (with a taurine concentration of 1.0 mM), compared to 46.70% in the control group.

[0057] Medium change treatment: Discard 880 μL of the mixed cells and add 1 mL of freshly prepared culture medium. Perform medium change treatment on days 9 and 11, discarding 950 μL of the mixed cells and adding 1 mL of freshly prepared culture medium. Perform cell counting and flow cytometry on day 13, and cell killing assay on day 14.

[0058] Preparation of effector cells: NK cells cultured to day 14 were centrifuged at 300×g for 5 min at room temperature. The liquid in the wells was then aspirated using a pipette (10 mL). 1 mL of fresh culture medium was added (45 mL RPMI 1640 basal medium + 5 mL serum + 500 μL penicillin and streptomycin + 500 μL NEAA). The cells were then resuspended, and 10 μL of the resuspended cells were used for cell counting. The cell count was then determined based on the NK cell ratio (CD3+) obtained from the flow cytometry results on day 13. - CD56 + Calculate the cell volume, then take out the NK cell volume (10,000 cells / well), and finally adjust the volume to 100 μL / well with the replaced culture medium.

[0059] Target cells (cancer cells): Remove A549 cells and observe cell growth under a microscope. Decan the culture medium, pipette out any remaining undecanted medium, add 1 mL of PBS buffer to wash, decan the medium, pipette out any remaining undecanted PBS buffer, then add 1 mL of trypsin for digestion. Incubate at 37°C with 5% CO2 for 2 min and observe the digestion. After digestion, add 3 mL of fresh culture medium to neutralize the digestion, then transfer to a 15 mL centrifuge tube, centrifuge at 300×g at room temperature for 5 min, decan the supernatant, resuspend in 1 mL of fresh culture medium, remove cells, count and calculate the cell count. Ensure a target cell quantity of 10,000 cells / well in 100 μL / well of cell suspension.

[0060] Control group: only 10,000 (100 μL) target cells were added, and then the volume was adjusted to 200 μL with a different culture medium.

[0061] Drug group: Effector cells and target cells (1:1) were mixed, centrifuged at 800×g at room temperature for 2 min, and co-cultured in a 37℃, 5% CO2 incubator for 5 h. After centrifugation at 300×g at room temperature for 5 min, the supernatant was discarded. To determine the cell viability: 2 μL of 7-AAD / well + 200 μL of APC-Annexin-V_Buffer / well + 1 μL of APC-Annexin-V / well were mixed and added to each well in 200 μL. The mixture was incubated at room temperature in the dark for 30 min, and the killing effect was detected by flow cytometry.

[0062] Flow cytometry results analysis on day 14 Figure 5 The results showed that the drug group performed better than the control group. In the NK cell percentage graph, CD3... - CD56 + The drug group with 1.0 mM taurine added to the ratio showed the best effect, CD3 - CD16 + The drug group with 0.4 mM taurine added showed the best effect. The drug group with 1.0 mM taurine added showed the best effect in the cell expansion map. The drug group with 0.1 mM taurine added showed the best effect in the killing effect map. Figure 6 The results showed that the overall scores of the drug groups with different concentrations (0.1~1.0mM) of taurine were better than those of the control group.

[0063] Example 2:

[0064] I. Experimental Materials

[0065] The basic NK cell culture medium was purchased from Shanghai Yuanpei Company. It was IMDM medium, catalog number L610KJ, with a specification of 500mL.

[0066] All reagents and kits are commercially available.

[0067] The taurine was purchased from MCE Company, catalog number HY-100803, specification 50mg. The purchased reagent was dissolved in 458μL of sterile water to obtain a 1mol / L stock solution for later use.

[0068] II. Experimental Procedures and Results

[0069] 1. Mononuclear cell isolation and flow cytometry detection

[0070] Peripheral blood was collected from healthy volunteers. After rinsing the blood collection bag containing the sample with 75% alcohol, the blood was transferred to a 50 mL centrifuge tube. The tube was centrifuged at 800×g at room temperature for 15 min. The lower layer was then removed and diluted with an equal volume of DPBS (Durbeco phosphate-buffered saline). This diluted solution was then slowly added along the wall of the centrifuge tube at a rate of 1–3 mL / min to the upper layer of the pre-added lymphocyte separation medium, ensuring a clear interface between the blood sample and the separation medium. The mixture was then centrifuged at 600×g at room temperature for 30 min, and the white cell layer (mononuclear cells) was collected. After washing twice with DPBS, cell counting was performed at a concentration of 2.0 × 10⁻⁶ cells / mL. 6 Cells were cryopreserved. The obtained cells were then analyzed by flow cytometry to obtain NK cells (CD3+). - CD56 + / CD3 - CD16 + )Proportion.

[0071] 2. Single-factor experiment to determine the effects of taurine on NK cell proliferation and killing.

[0072] This experiment used PBMC-16 (the 16th sample of peripheral blood mononuclear cells) for comparison. Cells cultured without the drug were set as the control group. Each group had three replicates. Taurine was added to the basal NK cell culture medium at concentrations of 0.4 mM, 0.8 mM, 1.0 mM, 2.0 mM, and 4.0 mM to obtain the drug-treated NK cell culture medium. PBMC-16 cells were initially cultured at a density of 1.5 million cells per cell in a 37°C, 5% CO2 incubator. The medium was initially added to 0.5 mL, with 0.5 mL added on day 3 and 1 mL added on day 5. Cell counting, flow cytometry analysis, and medium replacement were performed on day 7.

[0073] Cell counting: Remove the cells from the incubator, mix well, add 10 μL of cells to 10 μL of trypan blue for staining, and count using a cell counter. Detailed cell count and NK cell percentage changes are shown below. Figure 7 As shown, the cell count in the drug group with added 2.0 mM taurine was better than that in the control group. Figure 8The results showed that the drug groups with different concentrations (0.4~4.0mM) of taurine had a higher percentage of NK cells than the control group at both time points.

[0074] Flow cytometry: Take 60 μL of the mixed cells, centrifuge at 300×g for 5 min at room temperature, add 25 μL of the prepared flow cytometry antibody (PBS buffer + CD56 / CD3 / CD45 / CD16), incubate at 4℃ in the dark for 30 min, then wash with PBS buffer, centrifuge at 300×g for 5 min at room temperature, discard the supernatant, then add 200 μL of PBS buffer, mix well, incubate at room temperature in the dark for 10 min, and then detect NK cell expansion by flow cytometry. CD3 in mononuclear cells - CD56 + The proportion of NK cells in the phenotype was low, and after induction culture in NK medium containing taurine, CD3... - CD56 + The proportion of NK cells in the phenotype is significantly increased. Flow cytometry results show that, for example... Figure 9 As shown, compared with the control group on day 7, CD3 - CD56 + The phenotype rate increased to 57.48% in the drug group (37.19% phenotype concentration: 0.4 mM taurine) compared to the previous group. Figure 10 As shown, compared with the control group on day 13, CD3 - CD56 + The phenotype rate increased to 94.49% in the drug group (with a taurine concentration of 2.0 mM), compared to 89.83% in the control group.

[0075] Medium change: Discard 880 μL of the mixed cells and add 1 mL of freshly prepared culture medium. On day 11, perform cell counting and medium change, discard 950 μL of the mixed cells and add 1 mL of freshly prepared culture medium. On day 13, perform cell counting and flow cytometry analysis, and on day 14, perform cell killing assay.

[0076] Preparation of effector cells: NK cells cultured to day 14 were centrifuged at 300×g for 5 min at room temperature. The liquid in the wells was aspirated using a pipette (10 mL). 1 mL of fresh culture medium was added to the plate (45 mL RPMI 1640 basal medium + 5 mL serum + 500 μL penicillin and streptomycin + 500 μL NEAA). The cells were then resuspended, and 10 μL of the resuspended cells were used for cell counting. The cell count was then determined based on the NK cell ratio (CD3+) obtained from the flow cytometry results on day 13. - CD56 + Calculate the cell volume, then take out the NK cell volume (10,000 cells / well), and finally adjust the volume to 100 μL / well with the replaced culture medium.

[0077] Target cells (cancer cells): Remove A549 cells and observe cell growth under a microscope. Decant the culture medium, pipette out any remaining undecanted medium, add 1 mL of PBS buffer to wash, decant again, pipette out any remaining undecanted PBS buffer, then add 1 mL of trypsin for digestion. Incubate at 37°C with 5% CO2 for 2 min and observe the digestion. After digestion, add 3 mL of fresh culture medium to neutralize the digestion, then transfer to a 15 mL centrifuge tube and centrifuge at 300×g at room temperature for 5 min. Decant the supernatant, resuspend in 1 mL of fresh culture medium, remove the cells, count them, and calculate to ensure the target cell quantity (10,000 cells / well) is 100 μL / well.

[0078] Control group: only 10,000 (100 μL) target cells were added, and then the volume was adjusted to 200 μL with a different culture medium.

[0079] Drug group: Effector cells and target cells (1:1) were mixed, centrifuged at 800×g at room temperature for 2 min, and co-cultured in a 37℃, 5% CO2 incubator for 24 h. After centrifugation at 300×g at room temperature for 5 min, the supernatant was discarded. To determine the cell viability: 2 μL of 7-AAD / well + 200 μL of APC-Annexin-V Buffer / well + 1 μL of APC-Annexin-V / well were mixed and added to each well in 200 μL. The mixture was incubated at room temperature in the dark for 30 min, and the killing effect was detected by flow cytometry.

[0080] Flow cytometry results analysis on day 14, Figure 11 In the NK cell percentage diagram, CD3 - CD56 + The drug group with the addition of 0.4 mM taurine showed the best effect, CD3 - CD16 + The drug group with 0.4 mM taurine added showed the best effect in the cell expansion map. The drug group with 2.0 mM taurine added showed the best effect in the cell growth map. The drug group with 1.0 mM taurine added showed the best effect in the killing effect map. Figure 12 The results showed that the overall scores of the drug groups with different concentrations (0.4~4.0mM) of taurine were better than those of the control group.

[0081] Example 3:

[0082] I. Experimental Materials

[0083] The basic NK cell culture medium was purchased from Shanghai Yuanpei Company. It was IMDM medium, catalog number L610KJ, with a specification of 500mL.

[0084] All reagents and kits are commercially available.

[0085] The taurine was purchased from MCE Company, catalog number HY-100803, specification 50mg. The purchased reagent was dissolved in 458μL of sterile water to obtain a 1mol / L stock solution for later use.

[0086] II. Experimental Procedures and Results

[0087] 1. Mononuclear cell isolation and flow cytometry detection

[0088] Peripheral blood was collected from healthy volunteers. After rinsing the blood collection bag containing the sample with 75% alcohol, the sample was transferred to a 50mL centrifuge tube and centrifuged at 800×g at room temperature for 15 minutes. The lower layer was then collected and diluted with an equal volume of DPBS (Durbeco phosphate-buffered saline). The upper layer of lymphocyte separation medium was then slowly added, and the mixture was centrifuged at 600×g at room temperature for 30 minutes. The white cell layer was collected, yielding mononuclear cells. After washing twice with DPBS, cell counting was performed at a concentration of 2.0×10⁻⁶ cells / mL. 6 Cells were cryopreserved. The obtained cells were then analyzed by flow cytometry to obtain NK cells (CD3+). - CD56 + / CD3 - CD16 + )Proportion.

[0089] 2. Single-factor experiment to determine the effects of taurine on NK cell proliferation and killing.

[0090] This experiment used PBMC-18 (the 18th sample of peripheral blood mononuclear cells) for comparison. Cells cultured without the drug were set as the control group. Each group had three replicates. Taurine was added to the basal NK cell culture medium at concentrations of 0.4 mM, 0.8 mM, 1.0 mM, 2.0 mM, and 4.0 mM to obtain the drug-treated NK cell culture medium. PBMC-18 cells were initially cultured at a density of 1.5 million cells per cell in a 37°C, 5% CO2 incubator. The medium was initially added to 0.5 mL, with 0.5 mL added on day 3 and 1 mL added on day 5. Cell counting, flow cytometry analysis, and medium replacement were performed on day 7.

[0091] Cell counting: Remove the cells from the incubator, mix well, add 10 μL of cells to 10 μL of trypan blue for staining, and count using a cell counter. Detailed cell count and NK cell percentage changes are shown below. Figure 13 As shown, the cell counts in the drug groups with added 0.8 mM, 2.0 mM, and 4.0 mM concentrations of taurine were better than those in the control group. Figure 14 The results showed that the group receiving 4.0 mM taurine had significantly higher NK cell percentages than the control group at both time points.

[0092] Flow cytometry: Take 60 μL of the mixed cells, centrifuge at 300×g for 5 min at room temperature, add 25 μL of the prepared flow cytometry antibody (PBS buffer + CD56 / CD3 / CD45 / CD16), incubate at 4℃ in the dark for 30 min, then wash with PBS buffer, centrifuge at 300×g for 5 min at room temperature, discard the supernatant, then add 200 μL of PBS buffer, mix well, incubate at room temperature in the dark for 10 min, and then detect NK cell expansion by flow cytometry. CD3 in mononuclear cells - CD56 + The proportion of NK cells in the phenotype was low, and after induction culture in NK medium containing taurine, CD3... - CD56 + The proportion of NK cells in the phenotype is significantly increased. Flow cytometry results show that, for example... Figure 15 As shown, compared with the control group on day 7, CD3 - CD56 + The phenotype rate increased to 41.45% in the drug group (4.0 mM taurine concentration) compared to 32.57%. Figure 16 As shown, compared with the control group on day 13, CD3 - CD56 + The phenotype rate increased to 74.70% in the drug group (65.16% phenotype rate, 4.0 mM taurine concentration).

[0093] Medium change: Discard 880 μL of the mixed cells and add 1 mL of freshly prepared culture medium. On day 11, perform cell counting and medium change, discard 950 μL of the mixed cells and add 1 mL of freshly prepared culture medium. On day 13, perform cell counting and flow cytometry analysis, and on day 14, perform cell killing assay.

[0094] Preparation of effector cells: NK cells cultured to day 14 were centrifuged at 300×g for 5 min at room temperature. The liquid in the wells was aspirated using a pipette (10 mL). 1 mL of fresh culture medium was added to the plate (45 mL RPMI 1640 basal medium + 5 mL serum + 500 μL penicillin and streptomycin + 500 μL NEAA). The cells were then resuspended, and 10 μL of the resuspended cells were used for cell counting. The cell count was then determined based on the NK cell ratio (CD3+) obtained from the flow cytometry results on day 13. - CD56 + Calculate the cell volume, then take out the NK cell volume (10,000 cells / well), and finally adjust the volume to 100 μL / well with the replaced culture medium.

[0095] Target cells (cancer cells): Remove A549 cells and observe cell growth under a microscope. Decant the culture medium, pipette out any remaining undecanted medium, add 1 mL of PBS buffer to wash, decant again, pipette out any remaining undecanted PBS buffer, then add 1 mL of trypsin for digestion. Incubate at 37°C with 5% CO2 for 2 min and observe the digestion. After digestion, add 3 mL of fresh culture medium to neutralize the digestion, then transfer to a 15 mL centrifuge tube and centrifuge at 300×g at room temperature for 5 min. Decant the supernatant, resuspend in 1 mL of fresh culture medium, remove the cells, count them, and calculate to ensure the target cell quantity (10,000 cells / well) is 100 μL / well.

[0096] Control group: only 10,000 (100 μL) target cells were added, and then the volume was adjusted to 200 μL with a different culture medium.

[0097] Drug group: Effector cells and target cells (1:1) were mixed, centrifuged at 800×g at room temperature for 2 min, and co-cultured in a 37℃, 5% CO2 incubator for 24 h. After centrifugation at 300×g at room temperature for 5 min, the supernatant was discarded. To determine the cell viability: 2 μL 7-AAD / well + 200 μL APC-Annexin-V Buffer / well + 1 μL APC-Annexin-V / well were mixed and added to each well in 200 μL. The cells were incubated at room temperature in the dark for 30 min, and the killing effect was detected by flow cytometry.

[0098] Flow cytometry results analysis on day 14, Figure 17 In the NK cell percentage diagram, CD3 - CD56 + The drug group with the addition of 4.0 mM taurine showed the best effect, CD3 - CD16 + The drug group with a concentration of 4.0 mM taurine showed the best effect in the proportions. The drug group with a concentration of 4.0 mM taurine showed the best effect in the cell expansion diagram. The drug group with a concentration of 1.0 mM taurine showed the best effect in the killing effect diagram. Figure 18 The results showed that the overall scores of the drug groups with different concentrations (0.4~4.0mM) of taurine were better than those of the control group.

[0099] Comparative Example 1:

[0100] To verify the effectiveness of taurine, a comparative experiment was conducted using taurine. This comparative example was essentially the same as Example 1, except that taurine was used instead of taurine in this example. The concentrations of taurine added to the basal NK cell culture medium were set to 6.25 mM, 12.5 mM, 25 mM, 20 mM, 100 mM, and 200 mM to obtain the drug-treated NK cell culture medium. No replicates were used in Comparative Example 1.

[0101] The results are as follows Figure 19 , Figure 20 As shown, the drug groups supplemented with 6.25 μM and 25 μM taurine showed an advantage in cell count, but had a negative effect on the expansion of NK cell proportions; the proportions in all drug groups were significantly lower than those in the control group. In the final comprehensive scoring heatmap, as shown... Figure 21 As shown, the drug groups with added 6.25 μM and 25 μM taurine scored higher than the control group, but the cell-killing effect of the control group on day 13 was significantly lower than that of the drug group. In summary, adding taurine to the culture medium is less effective than adding taurine for cell proliferation and killing.

[0102] Based on the above experiments, the inventors also conducted tests on other basic NK cell culture media that do not contain taurine. The amount of taurine in the obtained NK culture media ranged from 0.1 to 4 mM, and all showed the same trend and effect. Basic NK cell culture media include, but are not limited to, one or more combinations of the following: DMEM medium, MEMα medium, DMEM / F12 medium, Gibco_CTS_NK-Xpander medium, FUJIFILM_PRIME-XV_NK Cell CDM medium, Miltenyi Biotec_NK MACS medium, R&D Systems_ExCellerate_Human NK Cell Expansion medium, STEMCELLImmunoCult NK Cell Base medium, CellGenix_NK Cell medium, Lonza_X-VIVO 10 medium, Lonza_X-VIVO 15 medium, Corning_KBM551, Corning_KBM581, Ekosei_NK serum-free medium, Youkang_NK cell serum-free medium, Dakowei_L500 lymphocyte serum-free medium, and Dakowei_N500 NK cell serum-free medium.

[0103] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An NK culture medium containing taurine, characterized in that: It consists of a basic NK cell culture medium and taurine; the basic NK cell culture medium is selected from RPMI_1640 medium or IMDM medium; When the basic NK cell culture medium is RPMI_1640 medium, the concentration of taurine is 0.8~1mM; when the basic NK cell culture medium is IMDM medium, the concentration of taurine is 1~2mM.

2. The NK culture medium containing taurine according to claim 1, characterized in that: The RPMI_1640 medium comprises the following raw materials in parts by weight: 10 parts glycine; 8.9 parts L-alanine; 200 parts L-arginine; 50 parts L-asparagine; 20 parts L-aspartic acid; 65 parts L-cysteine ​​dihydrochloride; 20 parts L-glutamic acid; and 15 parts L-histidine. 20 parts L-hydroxyproline; 50 parts L-isoleucine; 50 parts L-leucine; 40 parts L-lysine hydrochloride; 15 parts L-methionine; 15 parts L-phenylalanine; 20 parts L-proline; 30 parts L-serine; 20 parts L-threonine; 5 parts L-tryptophan; 29 parts L-tyrosine disodium salt; 20 parts L-valine; 2.5 parts ascorbate phosphate; 0.2 parts biotin; 3 parts choline chloride; 0.25 parts D-calcium pantothenate; 1 part folic acid; 1 part nicotinamide; 1 part para-aminobenzoic acid; 1 part pyridoxal hydrochloride; 0.2 parts riboflavin; 1 part thiamine hydrochloride; 0.005 parts vitamin B12; 35 parts inositol; 100 parts calcium nitrate tetrahydrate; 48.84 parts anhydrous magnesium sulfate; 400 parts potassium chloride; 2000 parts sodium bicarbonate; 6000 parts sodium chloride; 800 parts anhydrous disodium hydrogen phosphate; 0.874 parts zinc sulfate heptahydrate; 400 parts AlbuMAX® II serum-free culture medium supplement; 7.5 parts human transferrin; 10 parts recombinant insulin whole chain; 0.0003 parts ammonium vanadate; 0.00125 parts copper sulfate; 0.00005 parts manganese chloride; 0.005 parts sodium selenite; 2000 parts D-glucose; 1.9 parts ethanolamine; 1 part reduced glutathione; 110 parts sodium pyruvate.

3. The NK culture medium containing taurine according to claim 1, characterized in that: The IMDM culture medium comprises the following raw materials in parts by weight: 40 parts glycine; 33 parts L-alanine; 1083 parts L-alanyl-L-glutamine; 112 parts L-arginine hydrochloride; 38 parts L-asparagine; 40 parts L-aspartic acid; 93 parts L-cysteine; 100 parts L-glutamic acid; 56 parts L-histidine hydrochloride monohydrate; 140 parts L-isoleucine; 140 parts L-leucine; and 195 parts L-lysine hydrochloride. 40 parts L-methionine; 88 parts L-phenylalanine; 53 parts L-proline; 56 parts L-serine; 127 parts L-threonine; 21 parts L-tryptophan; 139 parts L-tyrosine disodium salt; 125 parts L-valine; 0.017 parts biotin; 5.3 parts choline chloride; 5.3 parts D-calcium pantothenate; 5.3 parts folic acid; 5.3 parts nicotinamide; 5.3 parts pyridoxal hydrochloride; 0.53 parts riboflavin; 5.3 parts thiamine hydrochloride; 0.017 parts vitamin B12; 9.6 parts inositol; 292 parts calcium chloride dihydrate; 267 parts magnesium sulfate heptahydrate; 440 parts potassium chloride; 0.10 parts potassium nitrate; 4032 parts sodium bicarbonate; 6000 parts sodium chloride; 188 parts sodium dihydrogen phosphate dihydrate; 0.023 parts sodium selenite pentahydrate; 6000 parts D-glucose; 7944 parts 4-hydroxyethylpiperazine ethanesulfonic acid; 147 parts sodium pyruvate.

4. The application of an NK culture medium containing taurine in enhancing NK cell proliferation and cytotoxic activity, characterized in that, NK cells are cultured using the NK culture medium according to any one of claims 1 to 3.

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