A method of inducing nk cells based on a porous material

Abstract

The application discloses a method for inducing NK cells based on a porous material. The method for inducing NK cells provided by the application is based on a device containing a mesh culture tank and porous gelatin sponge and a culture medium, and can efficiently induce iPSCs-NK. The method solves the problems that current differentiation culture technologies of iPSC-NK rely on feeder layer cells of animal origin, the differentiation efficiency is low, and the purity is insufficient and needs to be further sorted. The method has the potential for large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a method for inducing NK cells based on a porous material. BACKGROUND

[0002] NK cells (Nature killer cell, NK cell) are a kind of large granular lymphocytes discovered in the 1970s by a Swedish immunologist kiessling. The killing of tumor cells or infected cells by NK cells does not depend on the participation of antibodies, nor does it need antigen stimulation and sensitization; the recognition of target cells is not limited by major histocompatibility complex (MHC), so NK cells play a key role in the immune surveillance of viral infectious diseases and cancers. The killing mechanism of NK cells on tumor cells is mainly through the interaction of Fas (CD95) and FasL (CD95L), antibody-dependent cell-mediated cytotoxicity (ADCC), and the destruction of target cells by releasing cytotoxic effectors such as perforin and granzyme; at the same time, NK cells can secrete a large amount of cytokines such as tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) to promote the apoptosis of tumor cells. The important role of NK cells in tumor immunity makes NK cell adoptive immunotherapy a new strategy for treating tumors.

[0003] At present, the sources of NK cells used for immunotherapy mainly include peripheral blood mononuclear cells (PBMCs) and umbilical cord blood (UCB). PB-NK and UCB-NK cells are easy to obtain, but the number and purity of expanded NK cells are greatly affected by donors, and the products are usually heterogeneous.

[0004] Induced pluripotent stem cells (iPSCs) can differentiate into embryoid bodies (EBs) containing various germ layer sources under appropriate conditions in vitro, and further differentiate into functional cells related to treatment, which has broad clinical application prospects. Studies have shown that NK cells differentiated from iPSCs express a variety of NK receptors (NKG2D, TRAIL, CD16, etc.), and iPSC-NK can be produced from easily accessible sources such as fibroblasts or peripheral blood, maintain pluripotency during expansion, and be used for long-term storage. The advantages of the therapy can solve the supply chain bottleneck related to primary NK therapy and cell line NK therapy and have been widely concerned.

[0005] Although some progress has been made in the laboratory, there are still challenges in directly converting iPSCs into functionally mature NK cells, mainly manifested as: low efficiency of structural formation of NK precursor cells in the process of endothelial-hematopoietic transformation, low efficiency of induction and expansion of cells due to the inability of the generated NK cell precursors to be separated in time to the environment for NK cell expansion, and the difficulty of separating the target cells.

[0006] Therefore, more research and technical improvements are needed to address issues such as differentiation efficiency, cell stability, and functional performance, and the iPSCs technology and NK cell research have potential significance for the progress of immunotherapy and regenerative medicine. SUMMARY

[0007] To overcome the shortcomings of the prior art, the present application provides a high-efficiency iPSCs-NK induction method based on a porous material.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0009] The first aspect of the present application provides a device for inducing NK cells in vitro, comprising:

[0010] A culture tank and a water-absorbing suspended sponge material in the center thereof, the culture tank being a mesh sieve barrel-shaped culture tank.

[0011] Further, the culture tank is a mesh sieve barrel-shaped culture tank with a pore size of 40-100 μm.

[0012] Further, the culture tank is a mesh sieve barrel-shaped culture tank with a pore size of 40 μm.

[0013] Further, the water-absorbing suspended sponge material is selected from gelatin sponge.

[0014] Further, the device further comprises a vessel for placing the culture tank.

[0015] Further, the vessel is selected from a culture dish.

[0016] The second aspect of the present application provides a method for inducing NK cells, comprising culturing EBs in the device of the first aspect of the present application.

[0017] Further, the culture medium used for culturing EBs in the device of the first aspect of the present application is a fourth culture medium, which can induce differentiation of NK cell lineage.

[0018] Further, the fourth culture medium is a basic differentiation culture medium comprising SCF, VEGF, FGF, and IL.

[0019] Further, the medium used for culturing the EBs in the device of the first aspect of the application further comprises adding a fifth medium after inducing differentiation of the NK cell lineage, the fifth medium being a basal differentiation medium comprising VEGF, IL.

[0020] Further, the EBs are induced to differentiate from iPSCs.

[0021] Further, the medium for inducing iPSCs to differentiate into EBs comprises a first medium, a second medium and a third medium;

[0022] The first medium is a basal differentiation medium comprising GSK-3 inhibitor, BMP, activin A, SCF, VEGF, bFGF;

[0023] The second medium is a basal differentiation medium comprising GSK-3 inhibitor, BMP, TGF-β inhibitor, SCF, VEGF, FGF;

[0024] The third medium is a basal differentiation medium comprising BMP, SCF, IG II, FGF, VEGF.

[0025] Further, the GSK-3 inhibitor comprises a GSK-3a inhibitor or a GSK-3β inhibitor.

[0026] Further, the GSK-3 inhibitor is selected from a GSK-3β inhibitor.

[0027] Further, the GSK-3β inhibitor comprises one or more of CHIR99021, TD114-2, BIO, Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A014418, FRATide, Indirubin-3'-oxime, L803.

[0028] Further, the GSK-3β inhibitor is selected from CHIR99021.

[0029] Further, the BMP comprises one or more of BMP4, BMP2, BMP 6, BMP7, BMP15.

[0030] Further, the BMP is selected from BMP4.

[0031] Further, the FGF comprises one or more of FGF2, FGF7, FGF10.

[0032] Further, the FGF is selected from FGF2.

[0033] Further, the TGF-β inhibitor comprises one or more of SB-431542, A83-01, SB-505124, SB-525334, SD-208, LY-36494, SJN-2511.

[0034] Further, the TGF-β inhibitor is selected from SB-431542.

[0035] Further, the IL comprises one or more of IL2, IL15, IL17, IL6.

[0036] Further, the IL is selected from IL2, IL15.

[0037] Further, the concentration of CHIR99021 in the first medium is 0.5-1 μM.

[0038] Further, the concentration of CHIR99021 in the first medium is 0.5 μM.

[0039] Further, the concentration of CHIR99021 in the second medium is 3-6 μM.

[0040] Further, the concentration of CHIR99021 in the second medium is 3 μM.

[0041] Further, the concentration of BMP4 is 20-40 ng / mL.

[0042] Further, the concentration of BMP4 is 20 ng / mL.

[0043] Further, the concentration of activin A is 10 ng / mL.

[0044] Further, the concentration of SCF in the first medium and the second medium is 20 ng / mL.

[0045] Further, the concentration of SCF in the third medium is 50 ng / mL.

[0046] Further, the concentration of SCF in the fourth medium is 100 ng / mL.

[0047] Further, the concentration of VEGF in the first medium, the second medium and the third medium is 20 ng / mL.

[0048] Further, the concentration of VEGF in the fourth medium and the fifth medium is 50 ng / mL.

[0049] Further, the concentration of bFGF is 10-20 ng / mL.

[0050] Further, the concentration of bFGF is 10 ng / mL.

[0051] Further, the concentration of SB-431542 is 10 ng / mL.

[0052] Further, the concentration of FGF2 is 10-20 ng / mL.

[0053] Further, the concentration of FGF2 is 10 ng / mL.

[0054] Further, the concentration of IGII is 50 ng / mL.

[0055] Further, the concentration of IL2 is 10-20 ng / mL.

[0056] Further, the concentration of IL2 is 10 ng / mL.

[0057] Further, the concentration of IL15 is 10-20 ng / mL.

[0058] Further, the concentration of IL15 is 10 ng / mL.

[0059] Further, the first medium is a basic differentiation medium comprising 0.5 μM CHIR99021, 20 ng / mL BMP4, 10 ng / mL activin A, 20 ng / mL SCF, 20 ng / mL VEGF, 10 ng / mL bFGF;

[0060] The second medium is a basic differentiation medium comprising 3 μM CHIR99021, 20 ng / mL BMP4, 10 ng / mL SB-431542, 20 ng / mL SCF, 20 ng / mL VEGF, 10 ng / mL FGF2;

[0061] The third medium is a basic differentiation medium comprising 20 ng / mL BMP4, 20 ng / mL SCF, 50 ng / mL IGII, 10 ng / mL FGF2, 20 ng / mL VEGF;

[0062] The fourth medium is a basic differentiation medium comprising 100 ng / mL SCF, 50 ng / mL VEGF, 10 ng / mL FGF2, 10 ng / mL IL2, 10 ng / mL IL15;

[0063] The fifth medium is a basic differentiation medium comprising 50 ng / mL VEGF, 10 ng / mL IL2, 10 ng / mL IL15.

[0064] Further, the method comprises:

[0065] a) on day 1, iPSCs are induced by adding a first medium;

[0066] b) on day 2-3, iPSCs are induced by adding a second medium;

[0067] c) on day 4-8, iPSCs are induced by adding a third medium, and EBs are cystic blastula structures;

[0068] d) on day 8, EBs are transferred to the device of the first aspect of the application and supplemented with a fourth medium, and NK cell lineage differentiation is induced, with medium replacement every 2-3 days until day 26;

[0069] e) on day 26-32, a fifth medium is added, with medium replacement every 2-3 days.

[0070] Further, in steps d) and e), the medium is replaced every 3 days.

[0071] Further, step a) further comprises a step of digesting iPSCs.

[0072] Further, iPSCs are digested using Relesa digestion solution.

[0073] Further, step a) further comprises a step of washing iPSCs before digestion.

[0074] Further, iPSCs are washed using physiological saline.

[0075] Further, the cell confluency of iPSCs before washing is 80-90%.

[0076] Further, the cell confluency of iPSCs before washing is 80%.

[0077] Further, step a) further comprises a step of adding a culture solution after digestion.

[0078] Further, the culture solution is ncTarget culture solution.

[0079] Further, step a) further comprises a step of adjusting cell size after adding ncTarget culture solution.

[0080] Further, step a) further comprises a step of supplementing nutrients and growth factors after adjusting cell size.

[0081] Further, the growth factor is Y factor.

[0082] The third aspect of the application provides an NK cell population or derivative thereof, which is obtained by the method of the second aspect of the application.

[0083] The fourth aspect of the present application provides the use of the device of the first aspect of the present application in inducing NK cells.

[0084] The fifth aspect of the present application provides the use of the medium of the second aspect of the present application in inducing NK cells.

[0085] The sixth aspect of the present application provides the use of the NK cell population or its derivative of the third aspect of the present application in the preparation of a medicament for treating and / or preventing hematological diseases and / or autoimmune diseases and / or solid tumors.

[0086] Further, the medicament further comprises a pharmaceutically acceptable carrier.

[0087] Advantages and beneficial effects of the present application:

[0088] The method for inducing iPSCs-NK provided by the present application realizes the use of no other feeder layer cells by optimizing the induction conditions to form a hematopoietic precursor organoid containing NK cell-producing hemogenic endothelial cells and other necessary auxiliary cells required for hematopoietic differentiation. Further, by using a device containing a mesh culture tank and a porous gelatin sponge, the hematopoietic precursor organoid is made to grow along the porous structure of the sponge by culture with a specific culture medium, so that more endothelial-hematopoietic cell transformation sites are formed, and the yield of NK precursor cells is improved. Further, the generated NK precursor cells are separated from the birthplace under the action of gravity and gathered at the bottom of the culture dish, so that the automatic separation of NK cells is realized, and more specific conditions are provided for the expansion of NK cells. The method solves the problems of the current differentiation and culture technology of iPSC-NK, such as the dependence on animal-derived feeder layer cells, low differentiation efficiency, and the need for further sorting due to insufficient purity. The method of the present application is simple to operate and has the potential for large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 is a device schematic diagram, wherein 1 is a mesh with a pore size of 40 μm, 2 is a mesh support, and 3 is a gelatin sponge;

[0090] Figure 2 is an EBs culture to the 8th day result diagram;

[0091] Figure 3 is an EBs culture to the 17th day result diagram;

[0092] Figure 4 is an EBs culture to the 18th day result diagram;

[0093] Figure 5 is an EBs culture to the 23rd day result diagram;

[0094] Figure 6 is an EBs culture to the 32nd day result diagram;

[0095] Figure 7 is a flow detection result chart, wherein 7A is a CD56+ cell and CD8- cell flow detection result chart, and 7B is a CD56+ cell and CD45+ cell flow detection result chart. DETAILED DESCRIPTION

[0096] The following provides definitions of some terms used in the specification. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs.

[0097] The present application provides a device for inducing NK cells in vitro, comprising:

[0098] A culture tank and a water-absorbable suspended sponge material in the center thereof, the culture tank being a mesh barrel-shaped culture tank.

[0099] Specifically, the culture tank is a mesh barrel-shaped structure with a pore size of 40 μm, and contains a floating gelatin sponge with a hole structure. The method is suitable for the culture of iPSCs-NK obtained by rotating embryoid bodies (EBs). When used, the device is placed in a culture dish containing culture solution, the mesh design allows liquid to enter the culture tank, and EBs induced to a certain stage are placed on the surface. The device has the following four advantages: first, the gelatin sponge has a hollow structure, which provides a scaffold for EBs to adhere and produce a hematopoietic differentiation system of AGM-like hematopoietic organs, and provides a channel for endothelial-hematopoietic transformation to allow hematopoietic stem and progenitor cells to migrate to the next step. Second, the gelatin sponge is water-absorbing and floats, allowing EBs to be cultured for gas-liquid exchange, maintaining the matrix components (including endothelial cells) and supporting the expansion of AGM-derived hematopoietic progenitor cells, while the water-absorbing gelatin sponge becomes soft, providing a relatively suitable in vitro microenvironment for hematopoietic stem and progenitor cells. Third, it has a separation function, the gelatin sponge provides a suspension scaffold for adherent cells, the pore size structure provides a separation channel for suspended NK cells, allowing them to fall into the culture solution, making it easy to collect. At the same time, the mesh with a pore size of 40 μm can further filter the large-diameter cells that fall off, retaining large-diameter cells such as macrophages inside the mesh, achieving secondary filtration of NK cells and further improving NK purity. Fourth, the culture system of the present application is a pure factor method, which does not involve feeder cells and has high safety. Thus, the method can efficiently induce high-purity iPSCs-NK in vitro, realizing on-demand NK. The method is simple to operate and has the potential for large-scale industrial production.

[0100] In the present application, iPSCs, induced pluripotent stem cells, human induced pluripotent stem cells, induced pluripotent stem cells, can also be abbreviated as iPS cells or iPSCs, refer to a kind of pluripotent stem cells artificially prepared by introducing or contacting reprogramming factors, such as muscle cells, neurons, epidermal cells, etc., from non-pluripotent cells (usually adult somatic cells) or terminally differentiated cells (such as fibroblasts, hematopoietic cells).

[0101] In the present application, EBs, embryoid bodies, refer to blastoids or aggregates, which refer to homogeneous or heterogeneous cell clusters comprising differentiated cells, partially differentiated cells and / or pluripotent stem cells in suspension culture.

[0102] The present application provides a method for inducing NK cells, which comprises culturing EBs in the above-mentioned device.

[0103] The culture medium used for culturing EBs in the above-mentioned device is a fourth culture medium, which can induce differentiation of NK cell lineage.

[0104] The fourth culture medium is a basic differentiation medium comprising SCF, VEGF, FGF and IL.

[0105] The culture medium used for culturing EBs in the above-mentioned device further comprises a fifth culture medium added after inducing differentiation of NK cell lineage, and the fifth culture medium is a basic differentiation medium comprising VEGF and IL.

[0106] The EBs are induced to differentiate from iPSCs, and the culture medium for inducing iPSCs to differentiate into EBs comprises a first culture medium, a second culture medium and a third culture medium.

[0107] The first culture medium is a basic differentiation medium comprising GSK-3 inhibitor, BMP, activin A, SCF, VEGF and bFGF; the second culture medium is a basic differentiation medium comprising GSK-3 inhibitor, BMP, TGF-β inhibitor, SCF, VEGF and FGF; and the third culture medium is a basic differentiation medium comprising BMP, SCF, IGII, FGF and VEGF.

[0108] In the present application, GSK-3 inhibitor includes but is not limited to GSK-3α inhibitor or GSK-3β inhibitor.

[0109] In a preferred embodiment of the present application, the GSK-3 inhibitor is selected from GSK-3β inhibitor.

[0110] Examples of GSK-3 inhibitors include, but are not limited to, CHIR99021, TD114-2, BIO (6-bromoindirubin-30-acetone oxime), Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A 014418, FRATide, Indirubin-3'-oxime, or L803.

[0111] In a specific embodiment of the present application, the GSK-3 inhibitor is selected from CHIR99021.

[0112] In the present application, BMP (bone morphogeneitc protein) includes, but is not limited to, BMP4, BMP2, BMP 6, BMP7, BMP15.

[0113] In a specific embodiment of the present application, the BMP is selected from BMP4.

[0114] In the present application, TGF-β (transforming growth factor-β) inhibitors include, but are not limited to, SB-431542, A83-01, SB-505124, SB-525334, SD-208, LY-36494, SJN-2511.

[0115] In a specific embodiment of the present application, the TGF-β inhibitor is selected from SB-431542.

[0116] The culture medium of the present application can further comprise at least one amino acid. The amino acid includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof.

[0117] The culture medium of the present application can further comprise at least one vitamin. The vitamin includes, but is not limited to, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal / pyridoxal phosphate / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), ergocalciferol (vitamin D2), DL-alpha-tocopherol (vitamin E), biotin (vitamin H), menaquinone (vitamin K).

[0118] The medium of the present application can further comprise at least one inorganic salt. The inorganic salt includes, but is not limited to, salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. The salts are generally used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specifically, the salts can be exemplified by CaCl2, CuSO4.5H2O, Fe(NO3)3.9H2O, FeSO4.7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4.H2O, and ZnSO4.7H2O.

[0119] The medium of the present application can further comprise at least one sugar that can be a carbon energy source. The sugar includes glucose, galactose, mannose, fructose, and the like. Among them, as the sugar, glucose is preferred, and D-glucose (dextrose) is more preferred.

[0120] The medium of the present application can further comprise at least one trace element. The trace element includes barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.

[0121] The present application provides use of the above-mentioned NK cell population or derivative thereof in the preparation of a medicament for treating and / or preventing a hematological disease and / or an autoimmune disease and / or a solid tumor.

[0122] In the present application, treating and / or preventing means preventing, reversing, alleviating, inhibiting the progression of a disorder or condition to which the term applies or one or more symptoms of such disorder or condition, treating a disease or condition includes ameliorating at least one symptom of a particular disease or condition, even if the underlying pathophysiology is not affected, for example, treating and / or preventing a hematological disease in the present application includes one or more of: (1) preventing the occurrence of a hematological disease; (2) inhibiting the development of a hematological disease; (3) curing a hematological disease; (4) alleviating the symptoms associated with a hematological disease; (5) reducing the severity of a hematological disease; (6) preventing the recurrence of a hematological disease.

[0123] In the present application, the hematological disease includes, but is not limited to, chronic myelocytic leukemia, acute myelocytic 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, amegakaryocytic thrombocytopenia.

[0124] The autoimmune disease according to the present application refers to a disease resulting from an inappropriate immune response in a subject against substances and tissues normally present in the body. In other words, the immune system mistakenly attacks the body's own cells as if they were pathogens. This can be limited to certain tissues (e.g., in autoimmune thyroiditis) or involve specific tissues at different sites (e.g., Goodpasture's disease, which can affect the basement membranes of both the lungs and the kidneys). Treating autoimmune diseases generally uses immunosuppressive agents, e.g., drugs that reduce the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid disease, arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, anti-phospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, lyme arthritis, Guillain-Barre syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

[0125] The solid tumor according to the present application encompasses any type of cancer, in particular, the solid tumor includes, but is not limited to, cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, transitional cell carcinoma of the bladder, leukemia, brain tumor, stomach cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female genital tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma.

[0126] The medicament also includes a pharmaceutically acceptable carrier.

[0127] In the present application, the pharmaceutically acceptable carrier refers to a non-toxic material that does not interfere with the effect of the medicament of the present application or the biological activity of the medicament of the present application. Formulating a pharmaceutically active ingredient with a pharmaceutically acceptable carrier is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and any later edition). Non-limiting examples of pharmaceutically acceptable carriers include: salts (e.g., acid salts / anionic salts, base salts / cationic salts), excipients, buffers, diluents, solubilizers, tonicity adjusting agents, surfactants, preservatives, isotonic agents, flavoring agents, coloring agents, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the medicament of the present application.

[0128] In some embodiments of the application, the pharmaceutically acceptable carrier includes an acid / base salt. Non-limiting examples of acid / base salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide.

[0129] In some embodiments of the application, the pharmaceutically acceptable carrier includes an acid / base salt. Non-limiting examples of acid / base salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide.

[0130] In some embodiments of the application, the pharmaceutically acceptable carrier includes a buffer. Non-limiting examples of buffers include, but are not limited to, arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, triazine, and tris(hydroxymethyl)aminomethane, and mixtures thereof.

[0131] In some embodiments of the application, the pharmaceutically acceptable carrier includes a preservative. Non-limiting examples of preservatives include, but are not limited to, benzalkonium chloride, benzoic acid, benzyl alcohol, bromonitropropanediol, butylparaben, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethylparaben, imidurea, methylparaben, phenol, 2-phenoxyethanol, 2-phenylethanol, propylparaben, sodium dehydroacetate, thiomersal, and mixtures thereof.

[0132] In some embodiments of the application, the pharmaceutically acceptable carrier includes an isotonic agent. Non-limiting examples of isotonic agents include, but are not limited to, amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), sugar alcohols (such as glycerol, 1,2-propanediol, propylene glycol), 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (e.g., PEG 400), and mixtures thereof. Another example of an isotonic agent includes a sugar. Non-limiting examples of a sugar can be a monosaccharide, a disaccharide, or a polysaccharide, or a water-soluble dextran, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, where the term "sugar alcohol" is defined as a C(4-8) hydrocarbon with at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, hexanehexol, xylitol, and arabitol. A medicament comprising each of the isotonic agents listed in this paragraph constitutes alternative embodiments of the application.

[0133] In some embodiments of the application, the pharmaceutically acceptable carrier includes a chelating agent. Non-limiting examples of chelating agents include, but are not limited to, salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof.

[0134] In some embodiments of the application, the pharmaceutically acceptable carrier includes a stabilizer. Non-limiting examples of stabilizers include carboxy- / hydroxy cellulose and derivatives thereof (such as HPC, HPC-SL, HPC-L, and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycols (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as thioglycerol or mercaptoacetic acid.

[0135] In some embodiments of the application, the pharmaceutically acceptable carrier includes a lubricant. Lubricants can be used to prevent sticking of the dosage form to roller, die, and punch surfaces, and to reduce interparticulate friction. Lubricants can also facilitate ejection of the dosage form from the die cavity and improve granulation flow during processing. Examples of suitable lubricants include, but are not limited to, magnesium stearate, glyceryl behenate, calcium stearate, zinc stearate, stearic acid, silicon dioxide, talc, polyethylene glycol, mineral oil, carnauba wax, palmitic acid, sodium stearyl fumarate, sodium lauryl sulfate, glyceryl palmitostearate, myristic acid, and hydrogenated vegetable oils and fats, and other known lubricants, and / or mixtures of two or more thereof. In one embodiment, if present, the lubricant for granulation of the raw materials is magnesium stearate.

[0136] In some embodiments of the application, the pharmaceutically acceptable carrier comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term surfactant refers to any molecule or ion composed of a water-soluble portion (hydrophilic) and a fat-soluble and portion (lipophilic). For example, the surfactant is selected from the group consisting of: anionic surfactants, cationic surfactants, non-ionic surfactants, and / or zwitterionic surfactants.

[0137] The application is further illustrated by the following specific examples. It is to be understood that the particular examples described herein are included by way of illustration only and not as limitations of the application. The main features of the application can be used in various embodiments without departing from the scope of the application.

[0138] Example

[0139] 1. Experimental materials

[0140] The specific experimental materials are shown in Table 1.

[0141] Table 1 Experimental materials

[0142]

[0143]

[0144] 2. Experimental methods

[0145] I. Preparation of required solutions

[0146] 1. Preparation of 1.5% methylcellulose solution: weigh 1.25 g of methylcellulose powder, add 25 mL of sterile water, and vortex well until the powder is completely dissolved.

[0147] 2. Preparation of 2.5% polyvinyl alcohol solution: weigh 1.25 g of polyvinyl alcohol powder, add 25 mL of sterile water, and vortex well to swell, then heat melt in a 75°C water bath until completely dissolved.

[0148] 3. Preparation of 5% ascorbic acid-2-phosphate solution: weigh 1.25 g of ascorbic acid-2-phosphate powder, add 25 mL of sterile water, and vortex well until the powder is completely dissolved.

[0149] II. Preparation of basal differentiation medium

[0150] DMEM / F12 with 0.1% human blood albumin, 0.1% methylcellulose, 0.1% polyvinyl alcohol, 0.1% ascorbic acid-2-phosphate, 1x ITS EAF blood-free cell culture media supplement, 100 ng / mL linoleic and linolenic acid, 1x synthetic cholesterol, 22 nM 2-mercaptoethanol, 4% protein-free hybridoma mix II, 1x GlutaMAX.

[0151] III. iPSC balling and Nk induction differentiation

[0152] 1. Take out 80% confluent iPSCs, use a Pasteur pipette to aspirate the culture solution in the culture dish, and wash once with 2 mL of normal saline, and add 200 μL of Relesa digestion solution.

[0153] 2. Place in a 37°C, 5% CO2 constant oxygen incubator, and digest for 4 min.

[0154] 3. Immediately take out the culture dish after digestion is complete, and aspirate the Relesa digestion solution.

[0155] 4. Add 2 mL of ncTarget culture solution, and collect the cells by gently blowing. Try not to generate bubbles during the blowing process.

[0156] 5. Transfer all the cells to a 15 mL centrifuge tube, and adjust the size of the cell clumps by blowing several times with a Pasteur pipette.

[0157] 6. Blow the cells to a fine sand turbidity, and transfer most of the cells to a prepared 6-well plate. The volume of the culture solution in each well is 3 mL. If it is insufficient, supplement to 3 mL, and add 10 mM Y factor 6 μL.

[0158] 7. Place the 6-well plate in a 3D shaker at a speed of 65 r / min. After the balling is successful, add the corresponding culture medium for induction differentiation, and adjust the speed to 100 r / min.

[0159] 8. On day 1, add the basic differentiation medium containing 0.5 μM CHIR99021, 20 ng / mL BMP4, 10 ng / mL activin A, 20 ng / mL SCF, 20 ng / mL VEGF, and 10 ng / mL bFGF.

[0160] 9. On day 2-3, add the basic differentiation medium containing 3 μΜ CHIR99021, 20 ng / mL BMP4, 10 ng / mL SB-431542, 20 ng / mL SCF, 20 ng / mL VEGF, 10 ng / mL FGF2.

[0161] 10. On day 4-8, add the basic differentiation medium containing 20 ng / mL BMP4, 50 ng / mL SCF, 50 ng / mL IGII, 10 ng / mL FGF2, 20 ng / mL VEGF, and the EBs are cystic blastocyst structures.

[0162] 11. After day 8, transfer the cell spheres to the device for culture, and at this stage, add the basic differentiation medium containing 100 ng / mL SCF, 50 ng / mL VEGF, 10 ng / mL FGF2, 10 ng / mL IL2, and 10 ng / mL IL15 to induce differentiation of the NK lineage, and replace the medium every 3 days until day 26.

[0163] 12. On day 26-32, add the basic differentiation medium containing 50 ng / mL VEGF, 10 ng / mL IL2, and 10 ng / mL IL15, and replace the medium every 3 days. On day 32, collect the cells for flow cytometry analysis.

[0164] 3. Experimental results

[0165] The device of the present application is shown in Figure 1 , and the method is used under the condition of no feeder cells, and the EBs are induced to differentiate to completion on day 9, and can be placed on a gelatin sponge for culture Figure 2 . On day 17, the morphology of NK cells can be observed under a microscope Figure 3 , on day 18, the NK cells in the culture begin to aggregate into NK colonies Figure 4 , on day 23, the number of NK cells in the culture gradually increases Figure 5 , on day 32, the number of NK cells generated is basically saturated, and the NK colonies are well formed Figure 6 , and within 32 days, NK cells with a purity of 82.8% can be obtained, and about 100 million iPSCs can output about 1 billion iNK cells Figure 7 .

[0166] The above examples are only used to understand the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A method for inducing NK cells, characterized in that, The method includes culturing EBs in a device; The device includes: The culture tank and the water-absorbing suspended sponge material located in its center, wherein the culture tank is a culture tank with a mesh screen barrel structure; The culture tank is a mesh screen barrel-shaped culture tank with a pore size of 40-100μm; The water-absorbing and suspendable sponge material is selected from gelatin sponge; The device also includes a vessel for holding the culture tank; The method includes: a) On day 1, the first culture medium was added to the iPSCs for induction culture; b) On days 2-3, add the second culture medium for induction culture; c) On days 4-8, the third culture medium was added for induction culture, and the EBs were blastocyst structures; d) On day 8, EBs cell spheres were transferred to the device and supplemented with the fourth culture medium to induce NK cell lineage differentiation. The medium was changed every 2-3 days and cultured for 26 days. e) Add the fifth culture medium on days 26-32, and change the medium every 2-3 days; The fourth culture medium is a basic differentiation medium containing SCF, VEGF, FGF and IL; The fifth culture medium is a basic differentiation medium containing VEGF and IL; The first culture medium is a basal differentiation medium containing GSK-3 inhibitor, BMP, activin A, SCF, VEGF and bFGF; The second culture medium is a basic differentiation medium containing GSK-3 inhibitor, BMP, TGF-β inhibitor, SCF, VEGF and FGF; The third culture medium is a basic differentiation medium containing BMP, SCF, IGII, FGF and VEGF; GSK-3 inhibitors are selected from GSK-3β inhibitors; The GSK-3β inhibitor is selected from CHIR99021; The BMP is selected from BMP4; The FGF is selected from FGF2; The TGF-β inhibitor is selected from SB-431542; IL is selected from IL2 and IL15; The concentration of CHIR99021 in the first culture medium was 0.5~1 μM; The concentration of CHIR99021 in the second culture medium was 3-6 μM; The concentration of BMP4 is 20~40 ng / mL; The concentration of activin A was 10 ng / mL; The concentration of SCF in the first and second culture media was 20 ng / mL; The concentration of SCF in the third culture medium was 50 ng / mL; The concentration of SCF in the fourth culture medium was 100 ng / mL; The concentration of VEGF in the first, second, and third culture media was 20 ng / mL; The concentration of VEGF in the fourth and fifth culture media was 50 ng / mL; The concentration of bFGF is 10~20 ng / mL; The concentration of SB-431542 was 10 ng / mL; The concentration of FGF2 is 10~20 ng / mL; The concentration of IGⅡ was 50 ng / mL; The concentration of IL2 was 10-20 ng / mL; The concentration of IL15 is 10~20 ng / mL.

2. The method according to claim 1, characterized in that, The culture tank is a barrel-shaped culture tank with a mesh screen and a pore size of 40μm.

3. The method according to claim 1, characterized in that, The vessel is selected from petri dishes.

4. The method according to claim 1, characterized in that, The concentration of CHIR99021 in the first culture medium was 0.5 μM.

5. The method according to claim 1, characterized in that, The concentration of CHIR99021 in the second culture medium was 3 μM.

6. The method according to claim 1, characterized in that, The concentration of BMP4 was 20 ng / mL.

7. The method according to claim 1, characterized in that, The concentration of bFGF was 10 ng / mL.

8. The method according to claim 1, characterized in that, The concentration of FGF2 was 10 ng / mL.

9. The method according to claim 1, characterized in that, The concentration of IL2 was 10 ng / mL.

10. The method according to claim 1, characterized in that, The concentration of IL15 was 10 ng / mL.

11. The method according to claim 1, characterized in that, In steps d) and e), the fluid should be changed every 3 days.

12. The method according to claim 1, characterized in that, Step a) also includes the step of digesting iPSCs.

13. The method according to claim 12, characterized in that, iPSCs were digested using Relesa digestive solution.

14. The method according to claim 12, characterized in that, Step a) also includes a step of cleaning iPSCs before digestion.

15. The method according to claim 14, characterized in that, Use saline solution to clean iPSCs.

16. The method according to claim 15, characterized in that, Before washing, the cell confluence of iPSCs was 80-90%.

17. The method according to claim 16, characterized in that, The cell confluence of iPSCs was 80% before washing.

18. The method according to claim 12, characterized in that, Step a) also includes the step of adding culture medium after digestion.

19. The method according to claim 18, characterized in that, The culture medium is ncTarget culture medium.

20. The method according to claim 19, characterized in that, Step a) also includes adjusting cell size after adding ncTarget culture medium.

21. The method according to claim 20, characterized in that, Step a) also includes replenishing the nutrient solution and growth factors after the cells have reached full size.

22. The method according to claim 21, characterized in that, The growth factor is the Y factor.

23. The use of the first, second, third, fourth, and fifth culture media in the method of claims 1-10 in inducing NK cells.

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