Method for culturing NK (Natural Killer) cells and application

Abstract

The invention discloses a method for culturing NK (Natural Killer) cells and application. According to the method, the hPSCs are rotationally cultured in the 3D culture device to spontaneously form the EB, then the mixed cellulose ester (MCE) is used for further induced differentiation to the NK cells, and more NK cells with high proportion can be obtained. The method does not depend on 96-well plates, special consumables or matrigel and other matrigels, is high in balling efficiency and convenient to operate, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for culturing NK cells and an application thereof. Background Art

[0002] Natural killer cells (NK) are important immune cells in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity and autoimmune diseases in some cases. As the first line of defense of the body's defense system, NK cells can not only play their main anti-tumor effect in the innate immune system, but also can secrete different cytokines and various chemokines in the early stage of the immune response to regulate the body's acquired immune response. They are indispensable effector cells for the body to exert immune effects.

[0003] However, the number of NK cells is small, accounting for only 10%-15% of lymphocytes in peripheral blood, and the content of NK in umbilical cord blood is even lower, only about 5%. The content of NK cells in normal human peripheral blood and umbilical cord blood is far from meeting the needs of clinical treatment. There are many methods for culturing NK cells, and the sources of cells are also diverse, but the clinical application of NK cells is limited by factors such as the cumbersome culture process, high cost, and low cell ratio. How to provide a NK cell culture method that is easy to operate, low in cost, and high in culture efficiency is currently a research hotspot. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a method for culturing NK cells and applications thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] A first aspect of the present invention provides a method for inducing NK cells, the method comprising promoting cell differentiation into NK cells using a filtration membrane.

[0007] Furthermore, the filter membrane is selected from MCE (mixed cellulose ester).

[0008] Furthermore, the cells used for differentiation into NK cells are EBs (embryonic bodies).

[0009] Furthermore, the EBs are EBs containing hematopoietic stem and progenitor cells.

[0010] Furthermore, the culture medium components used for differentiating EBs into EBs containing hematopoietic stem and progenitor cells include: basal medium, albumin, methylcellulose, polyvinyl alcohol, GlutaMAX, ascorbic acid-2-phosphate, linoleic acid, cholesterol, 2-mercaptoethanol, and protein-free hybridoma mixture II.

[0011] Furthermore, the basal culture medium is selected from IMDM / F12 culture medium.

[0012] Furthermore, the culture medium components further include supplement 1 containing CHIR99021 or supplement 2 containing SB-431542.

[0013] Furthermore, the supplement 1 also includes BMP4, SCF, VEGF, and bFGF.

[0014] Furthermore, the supplement 2 also includes SCF, VEGF, and IL-3.

[0015] Furthermore, the EBs are obtained by culturing stem cells.

[0016] Furthermore, the stem cells are selected from pluripotent stem cells.

[0017] Furthermore, the pluripotent stem cells are selected from human pluripotent stem cells.

[0018] Furthermore, human pluripotent stem cells were cultured in a 3D device to form EBs.

[0019] Furthermore, the culture components used for culturing EBs include ncTarget medium, Blebbistain, Y-27632, and Thiazovivin.

[0020] Furthermore, the pore size of the MCE is 0.22-10 μm.

[0021] Furthermore, the culture system used for differentiation into NK cells is a basal culture medium.

[0022] Furthermore, the basal culture medium is selected from one or more of RPMI 1640, X-VIVO and KBI 581.

[0023] Furthermore, the culture system also includes SCF, FLT3L, IL2, IL-7, and IL-15.

[0024] The second aspect of the present invention provides the use of a filtration membrane in promoting cell differentiation into NK cells.

[0025] Furthermore, the filter membrane is selected from MCE.

[0026] Furthermore, the pore size of the MCE is 0.22-10 μm.

[0027] Furthermore, the cells used for differentiation into NK cells are EBs.

[0028] Furthermore, the EBs are EBs containing hematopoietic stem and progenitor cells.

[0029] Furthermore, the EBs are obtained by culturing stem cells.

[0030] Furthermore, the stem cells are selected from pluripotent stem cells.

[0031] Furthermore, the pluripotent stem cells are selected from human pluripotent stem cells.

[0032] Furthermore, human pluripotent stem cells were cultured in a 3D device to form EBs.

[0033] Furthermore, the culture system used for differentiation into NK cells is a basal culture medium.

[0034] Furthermore, the basal culture medium is selected from one or more of RPMI 1640, X-VIVO and KBI 581.

[0035] The third aspect of the present invention provides a NK cell population, which is cultured by the method described in the first aspect of the present invention.

[0036] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises the NK cell population described in the third aspect of the present invention.

[0037] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0038] The fifth aspect of the present invention provides the use of the method described in the first aspect of the present invention in culturing NK cells.

[0039] The sixth aspect of the present invention provides the use of the NK cell population described in the third aspect of the present invention or the pharmaceutical composition described in the fourth aspect of the present invention in the preparation of a drug for preventing / treating autoimmune diseases, blood system diseases or cancer.

[0040] The seventh aspect of the present invention provides the use of the NK cell population described in the third aspect of the present invention or the pharmaceutical composition described in the fourth aspect of the present invention in the preparation of a product having a killing effect on tumor cells.

[0041] Furthermore, the tumor cells are selected from Eca-1 and TE-1 cells.

[0042] Advantages and beneficial effects of the present invention: The present application cultivates hPSCs in a 3D culture device to form EBs, and then uses mixed cellulose esters (MCE) to further induce differentiation into NK cells, so that a high proportion and more NK cells can be harvested. The method of the present application does not rely on 96-well plates, special consumables, or matrix gels such as matrigel, and has high sphering efficiency and is easy to operate. Compared with matrix gels such as vitronectin and matrigel, the membrane material does not require pre-plating, and is cheap and easy to use; compared with mouse stromal cells such as OP9-DLL4 and OP9-DLL1, the membrane material does not require pre-resuscitation of cells and does not introduce heterologous components; compared with tumor cell lines such as K562, the membrane material has no carcinogenic risk. In addition, the membrane material can be sterilized at high temperature, is safe and reliable, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a diagram of the efficiency of hematopoietic differentiation after human pluripotent stem cells are formed into EBs in a 3D culture device, wherein 1A is a 3D EB ball culture device, 1B is a microphotograph of EB balls in a 3D rotating device on days 0 and 10, and 1C is a flow cytometry result diagram of differentiated EB balls on days 4, 6, and 10; Figure 2 This is a schematic diagram of inducing human pluripotent stem cells to differentiate into NK cells; Figure 3 Figure 3A is a graph of the efficiency of differentiation of human pluripotent stem cells into NK cells after forming EB spheres and inoculating them on different membrane materials. Figure 3A is a CD56 and CD45 flow cytometry graph of hiPSCs forming EB spheres and differentiating them for 10 days and then inoculating them on different materials for further culture for 15 days. Figure 3B and 3C are graphs of the percentage and yield of CD56+CD45+ NK cells after hiPSCs forming EB spheres and differentiating them for 10 days and then inoculating them on different materials for further culture for 15 days. Figure 4 It is a diagram of the efficiency of induction differentiation of human pluripotent stem cells into NK cells after being formed into EB spheres and inoculated into MCE materials with different pore sizes, wherein 4A is a CD56 and CD45 flow cytometry detection diagram after hiPSCs are formed into EB spheres and differentiated for 10 days and then inoculated into MCE materials with different pore sizes and cultured for 15 days, 4B and 4C are CD56+CD45+ NK cell percentage and yield diagrams after hiPSCs are formed into EB spheres and differentiated for 10 days and then inoculated into MCE materials with different pore sizes and cultured for 15 days; Figure 5Figure 5 is a graph showing the efficiency of differentiation of human pluripotent stem cells into NK cells in different induced differentiation systems after being inoculated in MCE after forming EB spheres. 5A is a CD56 and CD45 flow cytometry graph after hiPSCs formed EB spheres for 10 days and then were inoculated in different basal culture media for 30 days of induction differentiation. 5B and 5C are graphs showing the percentage and yield of CD56+CD45+ NK cells after hiPSCs formed EB spheres for 10 days and then were inoculated in different basal culture media for 30 days of induction differentiation. Figure 6 Figure 6 is a diagram of the killing effect of the cells obtained after human pluripotent stem cells formed EB spheres and inoculated in MCE for 30 days to induce differentiation into NK cells, respectively, after co-culture with human esophageal cancer cells Eca-1 and TE-1, wherein, 6A is a diagram of the survival rate of Eca-1 cells obtained after human pluripotent stem cells formed EB spheres and inoculated or not inoculated in MCE for 30 days to induce differentiation into NK cells, respectively, and co-cultured with human esophageal cancer cells Eca-1 for 0 to 72 hours, 6B is a diagram of the survival rate of TE-1 cells obtained after human pluripotent stem cells formed EB spheres and inoculated or not inoculated in MCE for 30 days to induce differentiation into NK cells, respectively, and 6C is a microscopic photograph of the cells obtained after human pluripotent stem cells formed EB spheres and inoculated or not inoculated in MCE for 30 days to induce differentiation into NK cells, respectively, and co-cultured with human esophageal cancer cells Eca-1 for 0, 6 and 12 hours; Figure 7 This is the flow cytometric graph of NK cell functional subtype markers CD16, granzyme CD107a and interferon IFN-γ obtained on the 30th day after human pluripotent stem cells formed EB spheres and were inoculated into MCE and induced to differentiate into NK cells. DETAILED DESCRIPTION

[0044] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0045] The present invention provides a method for inducing NK cells, which comprises using a filtration membrane to promote cell differentiation into NK cells.

[0046] In some embodiments, the filter membrane includes but is not limited to mixed cellulose ester (MCE), polyethersulfone (PES), polytetrafluorethylene (PTFE), polyvinylidenefluoride (PVDF), fiber glass, and nylon 66.

[0047] In a specific embodiment, the filtration membrane is selected from mixed cellulose ester (MCE).

[0048] In some embodiments, the pore size of the mixed cellulose ester (MCE) is 0.22-10 μm, for example, 0.22 μm, 0.45 μm, 0.65 μm, 1 μm, 2 μm, 3 μm, 5 μm, 8 μm, or 10 μm.

[0049] In a specific embodiment, the pore size of the mixed cellulose ester (MCE) is 2 μm.

[0050] In some embodiments, the cells used for differentiation into NK cells are EBs (embryonic bodies) formed by 3D culture of human pluripotent stem cells, which are multicellular aggregates.

[0051] The EBs are obtained by culturing stem cells.

[0052] In some embodiments, the stem cells include pluripotent stem cells and tissue stem cells.

[0053] In a preferred embodiment, the stem cells are selected from pluripotent stem cells.

[0054] In a specific embodiment, the pluripotent stem cells are selected from human pluripotent stem cells.

[0055] Human pluripotent stem cells are placed in a 3D device to culture and form EBs. The 3D device is a device named as a bioreactor and a bioreactor device, and the application number is 201721202793.0.

[0056] The culture system used for differentiation into NK cells is the basal medium.

[0057] In some embodiments, the basal culture medium includes but is not limited to RPMI 1640 culture medium, DMEM / F12 culture medium, X-VIVO 15 culture medium, AMI V culture medium, and KBM581 culture medium.

[0058] In a preferred embodiment, the basal culture medium is selected from RPMI 1640 culture medium, X-VIVO 15 culture medium or KBM 581 culture medium.

[0059] The present invention provides a NK cell population, which is cultured by the above method.

[0060] In some embodiments, the NK cell population can be used in combination with other therapeutic agents according to actual needs.

[0061] In some embodiments, the therapeutic agent includes but is not limited to an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an antiangiogenic agent, an anti-estrogen, an anti-androgen, an immunomodulator.

[0062] The alkylating agents include bischloroethyl methylamine, chlorambucil, propranolol, phenylmustard, estramustine, cyclophosphamide, hexamethylmelamine, triamine thionyl, carmustine, streptozotocin, forteptin, cyclohexyl lomustine, busulfan, thiosulfan, improsulfan, dacarbazine, cisplatin, oxaliplatin, and carboplatin.

[0063] The antimetabolites include methotrexate, 5-fluorouracil, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, cytarabine, 6-mercaptopurine (6-MP), 6-mercaptoguanine (6-TG), 2-chlorodeoxyadenosine, 5-azacytidine, cladribine, deoxycoformycin, and pentostatin.

[0064] The anti-tumor antibiotics include doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone hydrochloride, dactinomycin, mithramycin, mithramycin, mitomycin C, bleomycin, and procarbazine.

[0065] The mitotic inhibitors include paclitaxel, docetaxel, vinblastine, vincristine, vinblastine, and vinorelbine.

[0066] The chromatin function inhibitors include topotecan, irinotecan, etoposide, etoposide phosphate, and teniposide.

[0067] The anti-angiogenic agents include apromazine, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, neovastat, BMS-275291, and thalidomide.

[0068] The antiestrogens include anastrozole, letrozole, tamoxifen, toremifene, raloxifene, droloxifene, odoxifene, and exemestane.

[0069] The antiandrogens include flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride, and cimetidine.

[0070] The immunomodulators include interferon, interleukin, tumor necrosis factor, cisozumab, roquine, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase, and thymosin preparations.

[0071] The present invention provides a pharmaceutical composition, which comprises the above-mentioned NK cell population.

[0072] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0073] In some embodiments, pharmaceutically acceptable refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals, such as humans, and do not abrogate the biological activity and properties of the administered composition.

[0074] Pharmaceutically acceptable carriers include any and all aqueous solvents (e.g., water, alcohol / water solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, absorption delaying agents, salts, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids and nutritional supplements, such similar materials and combinations thereof, which will be known to those of ordinary skill in the art. The pH and exact concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0075] The actual dosage of the active ingredient in the pharmaceutical composition (such as the above-mentioned NK cell population or its derivatives) should be determined according to a variety of relevant factors, including the severity of the disease to be treated, the route of administration, the patient's age, gender, and weight. Therefore, the above dosage should not limit the scope of protection of the present invention in any way.

[0076] Administration can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The NK cell populations or derivatives thereof, pharmaceutical compositions or biological preparations described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the NK cell populations, pharmaceutical compositions or biological preparations provided herein can be administered to patients by intradermal or subcutaneous injection. In another embodiment, the NK cell populations, pharmaceutical compositions or biological preparations provided herein are preferably administered by i.v. injection. NK cell populations, pharmaceutical compositions or biological preparations can be directly injected into the patient's site, lymph nodes or infection site.

[0077] The present invention provides the use of the above-mentioned NK cell population or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing / treating autoimmune diseases, blood system diseases or cancer.

[0078] In some embodiments, the autoimmune disease includes refractory rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, juvenile idiopathic arthritis, systemic sclerosis, Wegener's granulomatosis, antiphospholipid antibody syndrome, severe myasthenia gravis, Crohn's disease, type 1 diabetes, and severe combined immunodeficiency.

[0079] In some embodiments, the blood system disease includes chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, aplastic anemia, Fanconi anemia, thalassemia, sickle cell anemia, myelofibrosis, severe paroxysmal nocturnal hemoglobinuria, and amegakaryocytic thrombocytopenia.

[0080] In some embodiments, the cancer includes but is not limited to cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, skin squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, bladder transitional cell carcinoma, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.

[0081] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0082] Example

[0083] Culture of human pluripotent stem cells The human pluripotent stem cells used in this example are normally cultured in a human pluripotent stem cell maintenance medium, and the medium used is E8, TeSR, ncTarget or other similar medium. The medium used in this experiment is ncTarget.

[0084] EB formation iPSCs were cultured as described above and embryoid body formation experiments were performed when the confluence was 90%.

[0085] The pluripotent stem cells were dissociated into single cells using mechanical or enzymatic dissociation methods known in the art. The iPSCs were digested into a complete single-cell suspension using accutase digestion enzyme. The cells were concentrated to about 1-5 × 10 6 / mL density was inoculated in ncTarget medium. ROCK inhibitors or other small molecule compounds that are beneficial to EB formation, such as Blebbistatin, Y-27632, Thiazovivin or a combination thereof, may be added to the medium, with an effective concentration of at least or about 1, 2.5, 3.0, 4.5, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, to about 20 μM, or any concentration range therein. In this experiment, ncTarget medium was supplemented with a combination of 5 μM Blebbistain, 5 μM Y-27632 and 5 μM Thiazovivin. This cell suspension was placed in a 37°C incubator and cultured in a rotating manner on a 3D shaker. EBs of relatively uniform size and morphology can be formed on the second day.

[0086] Differentiation of EBs into hematopoietic stem and progenitor cells On the first day of differentiation, transfer the medium containing EBs to a centrifuge tube and allow the EBs to settle to the bottom of the tube (5-10 minutes). Carefully aspirate the supernatant of the medium in the tube and replace it with hematopoietic differentiation medium every 2 days. On the first day of differentiation, use hematopoietic differentiation medium containing the small molecule compound Wnt / β-catenin signaling pathway activator CHIR99021, the effective concentration of which is at least or about 0.5, 1, 2, 3, 5, 8, 10 μM, where the effective concentration of CHIR99021 is 5 μM. On days 3-10 of differentiation, use hematopoietic differentiation medium containing the small molecule compound TGF-β receptor kinase inhibitor SB-431542, the effective concentration of which is at least or about 0.5, 1, 2, 3, 5, 8, 10 μM, where the effective concentration of SB-431572 is 5 μM.

[0087] The formula of the hematopoietic differentiation medium and the detailed information of related supplements are as follows: 0.1% recombinant human albumin, 0.1% methylcellulose (Sigma-Aldrich), 0.1% polyvinyl alcohol (Sigma-Aldrich), 1× GlutaMAX, 1× ascorbic acid-2-phosphate (Sigma-Aldrich), linoleic acid (100 ng / ml; Sigma-Aldrich), synthetic cholesterol (2.2 µg / ml; Sigma-Aldrich), 2-mercaptoethanol (22 nM), and protein-free hybridoma cocktail II (4%) were mixed in IMDM / F12 medium.

[0088] The first stage hematopoietic differentiation medium supplement (day 1-2) includes: CHIR99021 (5 μM), BMP4 (20 ng / ml), SCF (20 ng / ml), VEGF (20 ng / ml) and bFGF (10 ng / ml).

[0089] Supplements in the second phase (days 3–10) included: SB-431542 (5 μM; MCE), SCF (20 ng / ml), VEGF (20 ng / ml), and IL-3 (5 ng / ml).

[0090] Differentiation of EBs containing hematopoietic stem cells into NK cells On day 10, EBs were transferred onto filter membranes containing different pore sizes. NK differentiation medium containing cytokines was changed every two days.

[0091] The formula of NK differentiation induction solution and detailed information of related supplements are as follows: The basal culture medium is one or a mixture of RPMI1640 (Corning), AIM V™ (Life Technologies), X-Vivo15 medium (Lonza), DMEM / F12 (Gibco), and KBM 581 (Corning).

[0092] From day 10 to day 30, SCF (100 ng / ml), FLT3L (10 ng / ml), IL2 (10 ng / ml; Peprotech), IL-7 (20 ng / ml), and IL-15 (10 ng / ml) were added.

[0093] NK cell killing activity In order to further confirm whether the NK cells cultured with and without MCE materials have killing activity, this experiment was conducted using the Incucyte live cell analysis system. 24 hours in advance, the attached red fluorescent tumor cells Eca-1 or TE-1 were inoculated in a 96-well plate at a density of 10,000 cells / well. The next day, the IncuCyte Caspase-3 / 7 Green Apoptosis Assay Reagent (Essen Bioscience, 4440) was diluted 1000 times and added to the 96-well plate inoculated with tumor cells. The NK cells obtained after inoculation and without MCE culture were added at a ratio of E:T=5:1, and monitored on the IncuCyte live cell analysis system, with images collected every 1 hour. The experiment was repeated three times independently.

[0094] The cytotoxicity of target cells was analyzed by quantifying the ratio of Caspase 3 / 7 (green fluorescence) cells within red fluorescence cells.

[0095] Experiment 1: Spontaneous formation of EBs by rotating hiPSCs in a 3D culture device and then inducing hematopoietic differentiation Through the 3D culture device developed by this laboratory (a bioreactor and bioreactor equipment, application number: 201721202793.0) Figure 1 A) hPSCs were rotated to form EBs spontaneously, and then further induced to differentiate into hematopoietic cells in this device ( Figure 1 B). Flow cytometry analysis revealed that CD34 was detected on day 4 of differentiation. - KDR + Mesodermal cells (37.5%), about 12.1% CD34 were detected on day 6 + KDR + Hemogenic endothelial cells, about 3.7% CD34 were detected on day 10 + CD43 + Hematopoietic stem cell precursors and 2.2% CD34 + CD45 + Hematopoietic stem cells ( Figure 1 C).

[0096] Experiment 2: hiPSCs formed into EBs and then seeded on 2μm pore size MCE material can improve the efficiency of NK cell differentiation hiPSCs were rotated and cultured in a 3D culture device to spontaneously form EBs for differentiation. On the 10th day, EBs were seeded on different membrane materials to compare the NK differentiation efficiency ( Figure 2 ).

[0097] In order to further optimize the materials for NK-induced differentiation of hiPSCs through 3D formation of EB spheres containing hematopoietic stem and progenitor cells, we screened common membrane materials, including mixed cellulose ester (MCE) with a pore size of 0.45μm, polyethersulfone (PES), polytetrafluoroethylene (PTFE), polyvinylidenefluoride (PVDF), fiber glass, nylon 66, etc. It was found that MCE has a better support effect on NK-induced differentiation ( Figure 3 ).

[0098] In order to further optimize the support effect of MCE on NK cells, the support effect of MCE with different pore sizes of 0.22, 0.45, 0.65, 1, 2, 3, 5, 8, and 10 μm on NK cell differentiation was compared. 2 μm MCE has a better support effect on hiPSCs further differentiation into NK cells through EB spheres ( Figure 4 ). Therefore, 2µm pore size mixed cellulose esters provide better support for hiPSC-NK cells.

[0099] Experiment 3: Screening of different basal culture media In order to further study whether this material is suitable for other NK induced differentiation systems, different basal culture media (RPMI 1640, DMEM / F12, X-VIVO 15, AMI V, KBM 581) were screened ( Figure 5 ). It was found that hiPSCs formed EB spheres and differentiated for 10 days before being inoculated on MCE materials. The above culture media could increase the NK-induced differentiation ratio, but more NK cells could be obtained when inoculated on MCE in RPMI 1640, X-VIVO15 and KBM 581.

[0100] Experiment 4: Killing activity of NK cells Killing experiments confirmed that the NK cells induced by the method of the present application had a good killing effect on esophageal cancer cells Eca-1 and TE-1 ( Figure 6 ), and the expression of NK cell functional subtypes CD16, granzyme CD107a and interferon IFN-γ was significantly increased ( Figure 7 ).

[0101] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for inducing NK cells, characterized in that: The method includes using a filtration membrane to promote differentiation of cells into NK cells; Preferably, the filtration membrane is selected from MCE.

2. The method according to claim 1, characterized in that: The cells used for differentiation into NK cells are EBs; Preferably, the EBs are EBs containing hematopoietic stem and progenitor cells; Preferably, the culture medium components used for differentiating EBs into EBs containing hematopoietic stem and progenitor cells include: basal medium, albumin, methylcellulose, polyvinyl alcohol, GlutaMAX, ascorbic acid-2-phosphate, linoleic acid, cholesterol, 2-mercaptoethanol, and protein-free hybridoma mixture II; Preferably, the basal culture medium is selected from IMDM / F12 culture medium; Preferably, the culture medium components further include supplement 1 containing CHIR99021 or supplement 2 containing SB-431542; Preferably, the supplement 1 further comprises BMP4, SCF, VEGF, and bFGF; Preferably, the supplement 2 further comprises SCF, VEGF, and IL-3; Preferably, the EBs are obtained by culturing stem cells; Preferably, the stem cells are selected from pluripotent stem cells; Preferably, the pluripotent stem cells are selected from human pluripotent stem cells; Preferably, human pluripotent stem cells are cultured in a 3D device to form EBs; Preferably, the culture components used for culturing EBs include ncTarget medium, Blebbistain, Y-27632, and Thiazovivin.

3. The method according to claim 1, characterized in that: The pore size of the MCE is 0.22-10 μm.

4. The method according to claim 1, characterized in that: The culture system used for differentiation into NK cells is the basal medium; Preferably, the basal culture medium is selected from one or more of RPMI 1640, X-VIVO 15 and KBM 581; Preferably, the culture system further comprises SCF, FLT3L, IL2, IL-7, and IL-15.

5. Application of filtration membrane in promoting cell differentiation into NK cells; Preferably, the filter membrane is selected from MCE; Preferably, the pore size of the MCE is 0.22-10 μm; Preferably, the cells used for differentiation into NK cells are EBs; Preferably, the EBs are EBs containing hematopoietic stem and progenitor cells; Preferably, the EBs are obtained by culturing stem cells; Preferably, the stem cells are selected from pluripotent stem cells; Preferably, the pluripotent stem cells are selected from human pluripotent stem cells; Preferably, human pluripotent stem cells are cultured in a 3D device to form EBs; Preferably, the culture system used for differentiation into NK cells is a basal medium; Preferably, the basal culture medium is selected from one or more of RPMI 1640, X-VIVO 15 and KBM 581.

6. A NK cell population, characterized in that: The NK cell population is obtained by culturing the method according to any one of claims 1 to 4.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the NK cell population according to claim 6; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. Use of the method according to any one of claims 1 to 4 in culturing NK cells.

9. Use of the NK cell population according to claim 6 or the pharmaceutical composition according to claim 7 in the preparation of a drug for preventing / treating autoimmune diseases, blood system diseases or cancer.

10. Use of the NK cell population according to claim 6 or the pharmaceutical composition according to claim 7 in preparing a product having a killing effect on tumor cells; Preferably, the tumor cells are selected from Eca-1 and TE-1 cells.

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