A method for culturing nk cells and uses thereof

Abstract

The application discloses a method for culturing NK cells and application thereof. The application can harvest high proportion and more NK cells by rotating culture of hPSCs in a 3D culture device to form EBs spontaneously, and then further inducing differentiation of the NK cells by using mixed cellulose ester (MCE). The method of the application does not depend on 96-well plates, special consumables or matrigel and the like matrix glue, has high sphere formation efficiency and is convenient to operate, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for culturing NK cells and its application. Background Technology

[0002] Natural killer (NK) cells are important immune cells in the body, involved not only in anti-tumor, anti-viral infection, and immune regulation, but also, in certain cases, in the development of hypersensitivity reactions and autoimmune diseases. As the first line of defense in the body's immune system, NK cells not only play a major role in anti-tumor effects within the innate immune system, but also secrete various cytokines and chemokines in the early stages of the immune response to regulate the body's adaptive immune response. They are indispensable effector cells for the body to exert its immune effects.

[0003] However, NK cells are relatively few in number, accounting for only 10%-15% of lymphocytes in peripheral blood, and even less in umbilical cord blood, at only about 5%. The NK cell content in normal human peripheral blood and umbilical cord blood is far from meeting the needs of clinical treatment. Various methods exist for NK cell culture, and cell sources are diverse, but their clinical application is limited by factors such as cumbersome culture processes, high costs, and low cell proportions. Developing a convenient, low-cost, and highly efficient NK cell culture method is currently a research hotspot. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method for culturing NK cells and its application.

[0005] To achieve the above objectives, 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 using a filter membrane to promote cell differentiation into NK cells.

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

[0008] Furthermore, the cells used to differentiate into NK cells are EB (embryonic bodies).

[0009] Furthermore, the EB is an EB containing hematopoietic stem and progenitor cells.

[0010] Furthermore, the culture medium components used for EB differentiation into EB 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 also 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 EB is obtained from stem cell culture.

[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 EB.

[0019] Furthermore, the culture components used to cultivate EB 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 to differentiate into NK cells is based on the 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] A second aspect of the invention provides the use of a filter 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 to differentiate into NK cells are EB cells.

[0028] Furthermore, the EB is an EB containing hematopoietic stem and progenitor cells.

[0029] Furthermore, the EB is obtained from stem cell culture.

[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 EB.

[0033] Furthermore, the culture system used to differentiate into NK cells is based on the culture medium.

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

[0035] A third aspect of the present invention provides a population of NK cells obtained by the method described in the first aspect of the present invention.

[0036] A fourth aspect of the present invention provides a pharmaceutical composition comprising 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 application of the method described in the first aspect of the present invention in the culture of 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 medicament for the prevention / treatment of autoimmune diseases, hematological 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:

[0043] This application achieves a high proportion and greater number of NK cells by culturing hPSCs into EBs in a 3D culture device and then further inducing differentiation into NK cells using mixed cellulose esters (MCE). The method described in this application does not rely on 96-well plates, special consumables, or matrix gels such as Matrigel. It boasts high spheroidization efficiency and ease of operation. Compared to matrix gels such as vitrinin and Matrigel, this membrane material does not require pre-plating and is inexpensive and convenient to use. Compared to murine stromal cells such as OP9-DLL4 and OP9-DLL1, this membrane material does not require pre-resuscitation of cells and does not introduce heterologous components. Compared to tumor cell lines such as K562, this membrane material poses no carcinogenic risk. Furthermore, this membrane material can be sterilized at high temperatures, making it safe, reliable, and with broad application prospects. Attached Figure Description

[0044] Figure 1 This is a graph showing the hematopoietic induction differentiation efficiency of human pluripotent stem cells after EB formation in a 3D culture device. Among them, 1A is the 3D EB sphere culture device, 1B is the microscopic images of EB spheres on days 0 and 10 in the 3D rotating device, and 1C is the flow cytometry results of differentiated EB spheres on days 4, 6, and 10.

[0045] Figure 2 This is a schematic diagram illustrating the induction of human pluripotent stem cells into NK cells;

[0046] Figure 3 The graphs show the efficiency of differentiation of human pluripotent stem cells into NK cells after they are seeded onto different membrane materials after forming EB spheres. Among them, 3A is the flow cytometry graph of CD56 and CD45 after hiPSCs differentiate into EB spheres for 10 days and are seeded onto different materials for 15 days of further culture. 3B and 3C are the percentage and yield of CD56+CD45+ NK cells after hiPSCs differentiate into EB spheres for 10 days and are seeded onto different materials for 15 days of further culture.

[0047] Figure 4 The graphs show the efficiency of human pluripotent stem cells (hiPSCs) in inducing differentiation into NK cells after being seeded into MCEs of different pore sizes. Among them, 4A is a flow cytometry graph of CD56 and CD45 after hiPSCs differentiated into EB spheres for 10 days and then seeded into MCE materials of different pore sizes for 15 days. 4B and 4C are graphs of the percentage and yield of CD56+CD45+ NK cells after hiPSCs differentiated into EB spheres for 10 days and then seeded into MCE materials of different pore sizes for 15 days.

[0048] Figure 5This chart shows the differentiation efficiency of human pluripotent stem cells (hiPSCs) into NK cells after being seeded into MCEs in different induction differentiation systems. 5A shows flow cytometry data of CD56 and CD45 after 10 days of differentiation into EB spheres and induction into NK cells for 30 days in different basal media. 5B and 5C show the percentage and yield of CD56+CD45+ NK cells after 10 days of differentiation into EB spheres and induction into NK cells for 30 days in different basal media.

[0049] Figure 6 These are images showing the killing effect of human esophageal cancer cells Eca-1 and TE-1 after 30 days of NK cell differentiation induced by human pluripotent stem cells (HSCs) after forming EB spheres and being seeded into MCE. Image 6A shows the Eca-1 cell survival rate after 0-72 hours of co-culturing with human esophageal cancer cells Eca-1 after 30 days of NK cell differentiation induced by human pluripotent stem cells (with or without MCE seeding). Image 6B shows the TE-1 cell survival rate after 0-72 hours of co-culturing with human esophageal cancer cells TE-1 after 30 days of NK cell differentiation induced by human pluripotent stem cells (with or without MCE seeding). Image 6C shows micrographs of human esophageal cancer cells Eca-1 after 0, 6, and 12 hours of co-culturing with human esophageal cancer cells Eca-1 after 30 days of NK cell differentiation induced by human pluripotent stem cells (with or without MCE seeding).

[0050] Figure 7 This is a flow cytometry plot of NK cell functional subtype markers CD16, granzyme CD107a, and interferon IFN-γ obtained on day 30 after human pluripotent stem cells were seeded into MCEs following the formation of EB spheres. Detailed Implementation

[0051] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, 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 pertains.

[0052] The present invention provides a method for inducing NK cells, the method comprising using a filter membrane to promote cell differentiation into NK cells.

[0053] In some embodiments, the filter membrane includes, but is not limited to, mixed cellulose ester (MCE), polyethersulfone (PES), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), fiber glass, and Nylon 66.

[0054] In a specific embodiment, the filter membrane is selected from mixed cellulose ester (MCE).

[0055] In some embodiments, the pore size of the mixed cellulose ester (MCE) is 0.22-10 μm, for example, it can be 0.22 μm, 0.45 μm, 0.65 μm, 1 μm, 2 μm, 3 μm, 5 μm, 8 μm, or 10 μm.

[0056] In a specific embodiment, the pore size of the mixed cellulose ester (MCE) is 2 μm.

[0057] In some implementations, the cells used to differentiate into NK cells are EBs (embryonic bodies) formed from 3D culture of human pluripotent stem cells, which are multicellular aggregates.

[0058] The EB was obtained from stem cell culture.

[0059] In some embodiments, the stem cells include pluripotent stem cells and tissue stem cells.

[0060] In a preferred embodiment, the stem cells are selected from pluripotent stem cells.

[0061] In a specific implementation, the pluripotent stem cells are selected from human pluripotent stem cells.

[0062] Human pluripotent stem cells are cultured in a 3D device to form EBs, the 3D device being the device disclosed in the application No. 201721202793.0 entitled "A Bioreactor and Bioreaction Equipment".

[0063] The culture system used for differentiation into NK cells is the basal culture medium.

[0064] In some embodiments, the basal culture medium includes, but is not limited to, RPMI 1640 medium, DMEM / F12 medium, X-VIVO 15 medium, AMI V medium, and KBM581 medium.

[0065] In a preferred embodiment, the basal culture medium is selected from RPMI 1640 medium, X-VIVO 15 medium or KBM 581 medium.

[0066] This invention provides a population of NK cells, which is obtained by the above-described method.

[0067] In some implementations, the NK cell population may be used in combination with other therapeutic agents as needed.

[0068] In some embodiments, the therapeutic agents include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, and immunomodulators.

[0069] The alkylating agents include dichloroethyl methylamine, chlorambucil, bromoperazine, pine oxychloride, phosphatidylcholine, cyclophosphamide, hexamethyl melamine, thiamethoxam, carbamate, streptozomycin, flotinib, cyclohexanenitrosourea, busulfan, sulfan, indomethacin, damethoxam, cisplatin, oxaliplatin, and carboplatin.

[0070] 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, deoxycofromycin, and pentostatin.

[0071] The antitumor antibiotics include doxorubicin, daunorubicin, methoxybenzoic acid, pentorubicin, mitoxantrone hydrochloride, cyproterone acetate, sclerosingmycin, sclerosingmycin, mitomycin C, bleomycin, and procarbazine.

[0072] The mitotic inhibitors include paclitaxel, taxanes, vincristine, vinblastine, vinblastine, and vinorelbine.

[0073] The chromatin function inhibitors include topotecan, irinotecan, etopoxetine, etopoxetine phosphate, and podophyllotoxin.

[0074] The anti-angiogenic agents include propylthiouracil, mamastastat, palmatal, pralinestat, tannostat, ilomastastat, CGS-27023A, neovalprosta, BMS-275291, and thalidomide.

[0075] The anti-estrogens include anatozol, letrozol, tamoxifen, toremifene, raloxifene, droloxifene, oldoxifen, and exemestane.

[0076] The anti-androgens include flutamide, nilumet, bicalutamide, spironolactone, cyproterone acetate, phenacetin, and cimetidine.

[0077] The immunomodulators include interferon, interleukin, tumor necrosis factor, cizole, roquinomeprazole, pidotimod, methoxypolyethylene glycol succinamide adenosine deaminase, and thymopeptide preparations.

[0078] The present invention provides a pharmaceutical composition comprising the above-described NK cell population.

[0079] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0080] In some embodiments, pharmaceutically acceptable means a molecular entity and composition that, when administered to animals such as humans, does not produce adverse, allergic, or other inappropriate reactions, and does not negate the biological activity and properties of the administered composition.

[0081] Pharmaceutically acceptable carriers include any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral media such as sodium chloride, Ringer's glucose, 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., antimicrobial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, absorption delay agents, salts, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluids and nutritional supplements, and similar materials and combinations thereof, as will be known to those skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to known parameters.

[0082] The actual dosage of the active ingredient in the pharmaceutical composition (such as the NK cell population or its derivatives described above) should be determined based on a variety of relevant factors, including the severity of the disease to be treated, the route of administration, the patient's age, sex, and weight. Therefore, the above dosage should not limit the scope of protection of this invention in any way.

[0083] Administration can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The NK cell populations or their derivatives, pharmaceutical compositions, or biological agents described in this application can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (i.v.), or intraperitoneally. In one embodiment, the NK cell populations, pharmaceutical compositions, or biological agents provided in this application can be administered to a patient via intradermal or subcutaneous injection. In another embodiment, the NK cell populations, pharmaceutical compositions, or biological agents provided in this application are preferably administered via i.v. injection. NK cell populations, pharmaceutical compositions, or biological agents can be directly injected into the disease site, lymph node, or site of infection.

[0084] This invention provides the use of the above-described NK cell population or the above-described pharmaceutical composition in the preparation of medicaments for the prevention / treatment of autoimmune diseases, hematological diseases, or cancer.

[0085] In some embodiments, the autoimmune diseases include refractory rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, juvenile idiopathic arthritis, systemic sclerosis, Wegener's granulomatosis, antiphospholipid syndrome, severe myasthenia gravis, Crohn's disease, type 1 diabetes, and severe combined immunodeficiency.

[0086] In some embodiments, the hematologic disorders include chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, myelodysplastic syndrome, aplastic anemia, Fanconi anemia, thalassemia, sickle cell anemia, myelofibrosis, severe paroxysmal nocturnal hemoglobinuria, and amegakaryocyte thrombocytopenic purpura.

[0087] In some embodiments, the cancers include, but are not limited to, cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, squamous cell carcinoma of the skin, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, transitional cell carcinoma of the bladder, 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, and glioma.

[0088] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0089] Example

[0090] Culture of human pluripotent stem cells

[0091] The human pluripotent stem cells used in this embodiment were cultured normally in human pluripotent stem cell maintenance medium, such as E8, TeSR, ncTarget, or other similar media. The medium used in this experiment was ncTarget.

[0092] EB formation

[0093] When iPSCs are cultured to 90% confluence using the method described above, embryoid formation experiments are performed.

[0094] Pluripotent stem cells (iPSCs) were dissociated into single cells using mechanical or enzymatic dissociation methods known in the art. The iPSCs were then digested into a complete single-cell suspension using accutase digestive enzymes, with the cells at a concentration of approximately 1–5 × 10⁻⁶ cells / cells. 6 Cells were seeded at a density of 1 / mL in ncTarget medium. ROCK inhibitors or other small molecule compounds that promote EB formation, such as Blebbistainin, Y-27632, Thiazovivin, or combinations thereof, can be added to the medium at effective concentrations 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, up to about 20 μM, or any concentration range thereof. In this experiment, 5 μM Blebbistain, 5 μM Y-27632, and 5 μM Thiazovivin were added to the ncTarget medium. This cell suspension was placed in a 3D shaker at 37°C for incubation. EBs of relatively uniform size and morphology formed on day 2.

[0095] Differentiation of EB into hematopoietic stem and progenitor cells

[0096] On day 1 of differentiation, transfer the culture medium containing EB to centrifuge tubes and allow the EB to settle to the bottom of the tube (5-10 minutes). Carefully aspirate the supernatant from the tubes and change the medium every 2 days using hematopoietic differentiation medium. On day 1 of differentiation, use hematopoietic differentiation medium containing the small molecule compound Wnt / β-catenin signaling pathway activator CHIR99021 at effective concentrations of at least or approximately 0.5, 1, 2, 3, 5, 8, or 10 μM. Here, the effective concentration of CHIR99021 is 5 μM. From day 3 to day 10 of differentiation, use hematopoietic differentiation medium containing the small molecule compound TGF-β receptor kinase inhibitor SB-431542 at effective concentrations of at least or approximately 0.5, 1, 2, 3, 5, 8, or 10 μM. Here, the effective concentration of SB-431572 is 5 μM.

[0097] The formulation of the hematopoietic induction differentiation medium and detailed information on related supplements are as follows:

[0098] 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 mixture II (4%) were mixed into IMDM / F12 medium.

[0099] The first-stage hematopoietic differentiation culture medium supplement (days 1-2) includes: CHIR99021 (5 μM), BMP4 (20 ng / ml), SCF (20 ng / ml), VEGF (20 ng / ml) and bFGF (10 ng / ml).

[0100] The second phase of supplementation (days 3-10) includes: SB-431542 (5 μM; MCE), SCF (20 ng / ml), VEGF (20 ng / ml) and IL-3 (5 ng / ml).

[0101] Differentiation of hematopoietic stem and progenitor cells (EB) into NK cells

[0102] On day 10, EB was transferred to a filter membrane containing different pore sizes. The NK differentiation medium containing cytokines was changed every two days.

[0103] The formulation of the NK-induced differentiation medium and detailed information on related supplements are as follows:

[0104] The basal culture media are one or a mixture of several of the following: RPMI1640 (Corning), AIM V™ (Life Technologies), X-Vivo15 medium (Lonza), DMEM / F12 (Gibco), and KBM 581 (Corning).

[0105] From day 10 to day 30, add SCF (100 ng / ml), FLT3L (10 ng / ml), IL2 (10 ng / ml; Peprotech), IL-7 (20 ng / ml), and IL-15 (10 ng / ml).

[0106] NK cell killing activity

[0107] To further confirm the cytotoxic activity of NK cells cultured with and without MCE seeding, the Incucyte live cell analysis system was used in this experiment. Adherent Eca-1 or TE-1 tumor cells expressing red fluorescence were seeded into 96-well plates at a density of 10,000 cells / well 24 hours prior to the experiment. The next day, IncuCyte Caspase-3 / 7 GreenApoptosis Assay Reagent (Essen Bioscience, 4440) diluted 1000-fold was added to the 96-well plates seeded with tumor cells. NK cells obtained after MCE seeding and those without seeding were added at an effector-to-target ratio of E:T = 5:1. Monitoring was performed on the IncuCyte live cell analysis system, with images acquired every 1 hour. Three independent biological replicates were used in the experiment.

[0108] The cytotoxicity of target cells was analyzed by quantifying the ratio of Caspase 3 / 7 (green fluorescent) cells in red fluorescent cells.

[0109] Experiment 1 involved rotating hiPSCs in a 3D culture device to spontaneously form EBs, followed by hematopoietic induction and differentiation.

[0110] The 3D culture device (a bioreactor and bioreaction equipment, application number: 201721202793.0) developed in this laboratory ( Figure 1 A) hPSCs were rotated and cultured to spontaneously form EBs, which were then further induced to differentiate into hematopoietic cells in the device. Figure 1 B). Flow cytometry analysis revealed the presence of CD34 on day 4 of differentiation. - KDR + Mesodermal cells (37.5%) showed approximately 12.1% CD34 on day 6. + KDR + Hematopoietic endothelial cells showed approximately 3.7% CD34 on day 10. + CD43 + Hematopoietic stem progenitor cells and 2.2% CD34 + CD45 + Hematopoietic stem cells ( Figure 1 C).

[0111] Experiment 2: Seeding hiPSCs into 2μm pore size MCE material after EB formation can improve NK cell differentiation efficiency.

[0112] hiPSCs were spontaneously induced to differentiate by rotating culture using a 3D culture device. On day 10, the EBs were seeded onto different membrane materials to compare the NK-induced differentiation efficiency. Figure 2 ).

[0113] To optimize the NK-induced differentiation material for hiPSCs through the 3D formation of EB spheres containing hematopoietic stem and progenitor cells, commonly used membrane materials were screened, including mixed cellulose ester (MCE) with a pore size of 0.45 μm, polyethersulfone (PES), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), fiber glass, and Nylon 66. MCE was found to provide better support for NK-induced differentiation. Figure 3 ).

[0114] To further optimize the support effect of MCE on NK cells, the effects of MCEs with different pore sizes (0.22, 0.45, 0.65, 1, 2, 3, 5, 8, and 10 μm) on NK cell differentiation induction were compared. A 2 µm MCE showed good support for the further NK cell differentiation of hiPSCs via EB spheres. Figure 4 Therefore, 2µm pore size mixed cellulose esters provide better support for hiPSC-NK cells.

[0115] Experiment 3: Screening different basal culture media

[0116] To further investigate whether this material is suitable for other NK-induced differentiation systems, different basal media (RPMI 1640, DMEM / F12, X-VIVO 15, AMI V, KBM 581) were screened. Figure 5 It was found that when hiPSCs were seeded onto MCE material 10 days after EB sphere differentiation, the NK-induced differentiation ratio was increased in all of the above-mentioned culture media. However, seeding onto MCE in RPMI 1640, X-VIVO15 and KBM 581 resulted in more NK cells.

[0117] Experiment 4: Killing Activity of NK Cells

[0118] Killing experiments confirmed that NK cells induced using the method described in this application have a good killing effect on esophageal cancer cells Eca-1 and TE-1. Figure 6 Furthermore, the expression of NK cell functional subtypes CD16, granzyme CD107a, and interferon IFN-γ was significantly enhanced. Figure 7 ).

[0119] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method of inducing NK cells, characterized by, The method comprises using a filter membrane to promote differentiation of cells into NK cells; The filter membrane is MCE; The cells used for differentiation into NK cells are embryoid bodies; The embryoid bodies are hematopoietic stem / progenitor cell-containing embryoid bodies; The embryoid bodies are obtained by culturing human pluripotent stem cells.

2. The method of claim 1, wherein, Components of the culture medium used for differentiation of the embryoid bodies into hematopoietic stem / progenitor cell-containing embryoid bodies include: basal medium, albumin, methyl cellulose, polyvinyl alcohol, GlutaMAX, ascorbic acid-2-phosphate, linoleic acid, cholesterol, 2-mercaptoethanol, and protein-free hybridoma mixture II.

3. The method of claim 2, wherein, The basal medium is selected from IMDM / F12 medium.

4. The method of claim 2, wherein, The components of the culture medium further include CHIR99021-containing supplement 1 or SB-431542-containing supplement 2.

5. The method of claim 4, wherein, The supplement 1 further includes BMP4, SCF, VEGF, and bFGF.

6. The method of claim 4, wherein, The supplement 2 further includes SCF, VEGF, and IL-3.

7. The method of claim 1, wherein, The human pluripotent stem cells are cultured in a 3D device to form embryoid bodies.

8. The method of claim 7, wherein, Culture components used for forming the embryoid bodies include ncTarget medium, Blebbistain, Y-27632, and Thiazovivin.

9. The method of claim 1, wherein, The pore size of the MCE is 0.22-10 μm.

10. The method of claim 1, wherein, The culture system used for differentiation into NK cells includes a basal medium.

11. The method of claim 10, wherein, The basal medium is selected from one or more of RPMI 1640, X-VIVO15, and KBM 581.

12. The method of claim 10, wherein, The culture system further includes SCF, FLT3L, IL2, IL-7, and IL-15.

13. Use of a filter membrane in promoting differentiation of cells into NK cells; The filter membrane is selected from MCE; The cells used for differentiation into NK cells are embryoid bodies; The embryoid bodies are hematopoietic stem / progenitor cell-containing embryoid bodies; The embryoid bodies are obtained by culturing human pluripotent stem cells.

14. Use according to claim 13, characterized in that, The pore size of the MCE is 0.22-10 μm.

15. The use according to claim 13, characterized in that, The human pluripotent stem cells are cultured in a 3D device to form embryoid bodies.

16. The use according to claim 13, characterized in that, The culture system used for differentiation into NK cells is a basal medium.

17. Use according to claim 16, characterized in that, The basal medium is selected from one or more of RPMI 1640, X-VIVO15, and KBM 581.

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