Method and kit for producing immune cell differentiated from stem cell

Abstract

To provide a method for producing an immune cell differentiated from a stem cell.SOLUTION: Provided is a method for producing an immune cell differentiated from a stem cell. The method includes: (a) a step of forming coating including a matrix on a surface; and (b) a step of culturing a stem cell on the coating in the presence of first cytokine combination, and differentiating the stem cell into an immune cell, where the immune cell includes a γδT-cell (gamma delta T cell, GDT cell). The matrix includes a vascular cell adhesion molecule (VCAM), an intercellular adhesion molecule (ICAM) and a δ-like ligand 4 (DLL4). The first cytokine combination includes a stem cell factor (SCF), a thrombopoietin (TPO), an Fms-like tyrosine kinase 3 ligand (Flt3 L) and an interleukin-7 (IL-7). Further, a source of the stem cell includes umbilical cord blood or an induced pluripotent stem cell (iPSC), and the stem cell is CD34 positive.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to the differentiation of stem cells, and particularly to methods and kits for producing immune cells differentiated from stem cells.

Background Art

[0002] Immune cell therapy is currently a new trend in cancer treatment. However, the culture and expansion technology using autologous immune cells often fails to achieve the expected treatment results due to the poor function of the patient's immune cells, and it is necessary to incur more costs for quality and process management, so most patients cannot afford it. Therefore, the development of high-quality allogeneic immune cells (allogenic therapy) will be an important direction in improving the efficacy of immune cell therapy.

[0003] Hematopoietic stem cells (HPSCs) are a population of stem cells that have the potential to differentiate into other blood cells in the blood. Only a small amount of hematopoietic stem cells is present in the whole blood of adults, but a larger amount is present in umbilical cord blood. Overseas research has shown that the utilization rate of umbilical cord blood per person is less than 1 / 10,000. Therefore, if an efficient in vitro methodology can be developed to differentiate hematopoietic stem cells into therapeutic immune cells, such as gamma delta T cells (GDT cells), NK cells, etc., the application range of stored umbilical cord blood can be expanded, and the development of the umbilical cord blood business can be promoted.

[0004] Currently, research shows that clinically significant situations are usually not directly related to gamma delta T cell therapy, regardless of in vivo proliferation, in vitro stimulated proliferation and infusion, or autologous or allogeneic blood transfusion. Therefore, gamma delta T cell-based cellular immunotherapy is clinically relatively safe. In addition, gamma delta T cell-based cellular immunotherapy usually does not need to include a lymphocyte depletion step. Therefore, gamma delta T cell-based cellular immunotherapy is currently one of the targets that should be focused on developing in cell therapy.

[0005] In the existing technology, the peripheral blood separation method is often used to obtain γδ T cells. However, the initial cell number of γδ T cells obtained by this method is small, and a sufficient therapeutic dose cannot be obtained without an in vitro amplification method.

[0006] Therefore, how to obtain a sufficient therapeutic dose of γδ T cells in a simple manner is the current urgent issue and the direction in which research should proceed. Summary of the Invention Problems to be Solved by the Invention

[0007] Currently, research has shown that the cellular immunotherapy of γδ T cells is relatively safe clinically. However, the peripheral blood separation method is often used to obtain γδ T cells. The initial cell number of γδ T cells obtained by this method is small, and a sufficient therapeutic dose cannot be obtained without an in vitro amplification method. The main problem of this disclosure is how to obtain a sufficient therapeutic dose of γδ T cells in a simple manner. Means for Solving the Problems

[0008] The present invention provides a method and a kit for producing immune cells (particularly γδ T cells) differentiated from stem cells.

[0009] That is, this disclosure may include the following contents, but is not limited thereto.

[0010] [1] A method for producing immune cells differentiated from stem cells, comprising: (a) forming a coating containing a matrix on the surface; (b) culturing stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells, wherein the immune cells include γδ T cells (gamma delta T cell, GDT cell), and the matrix is vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), delta-like ligand 4 (DLL4), and the combination of the first cytokine is stem cell factor (SCF), thrombopoietin (TPO), Fms-like tyrosine kinase 3 ligand (Flt3L), interleukin-7 (IL-7), and the source of the stem cells includes umbilical cord blood or induced pluripotent stem cells (iPSCs), and the stem cells are CD34 positive, a method.

[0011] [2] The coating is formed from a coating-forming solution, and the coating-forming solution contains the matrix, a method for producing immune cells differentiated from the stem cells according to [1].

[0012] [3] In the coating-forming solution, the concentration of the vascular cell adhesion molecule is 0.05 to 50 μg / mL, the concentration of the intercellular adhesion molecule is 0.05 to 50 μg / mL, and the concentration of the delta-like ligand 4 is 0.05 to 50 μg / mL, a method for producing immune cells differentiated from the stem cells according to [1].

[0013] [4] In step (b), the concentration of the stem cell factor is 1 to 500 ng / mL, the concentration of the thrombopoietin is 1 to 500 ng / mL, the concentration of the Fms-like tyrosine kinase 3 ligand is 1 to 500 ng / mL, and the concentration of the interleukin-7 is 1 to 500 ng / mL, a method for producing immune cells differentiated from the stem cells described in [1].

[0014] [5] In step (b), the stem cells are cultured at 35 to 37 °C for 7 to 42 days, a method for producing immune cells differentiated from the stem cells described in [1].

[0015] [6] Before step (b), further comprising the step of (b’) screening the stem cells from the source of the stem cells, a method for producing immune cells differentiated from the stem cells described in [1].

[0016] [7] Before step (b), further comprising the step of (b’’) amplifying the stem cells, a method for producing immune cells differentiated from the stem cells described in [1].

[0017] [8] Before step (b), (b’) the step of screening the stem cells from the source of the stem cells, and (b’’) the step of amplifying the stem cells after step (b’) further comprising a method for producing immune cells differentiated from the stem cells described in [1].

[0018] [9] The process of amplifying the stem cells is (b’’-1) including the step of culturing the stem cells in the presence of a combination of a second cytokine, a method for producing immune cells differentiated from the stem cells described in [7] or [8].

[0019]

[10] The combination of the second cytokine is stem cell factor, and thrombopoietin, and Fms-like tyrosine kinase 3 ligand, and Interleukin-3, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-6, and a method for producing immune cells differentiated from the stem cells described in [9]. Interleukin-6, and A method for producing immune cells differentiated from the stem cells according to [9], comprising:

[0020]

[11] In step (b''), the concentration of the stem cell factor is 1 to 500 ng / mL, the concentration of the thrombopoietin is 1 to 500 ng / mL, the concentration of the Fms-like tyrosine kinase 3 ligand is 1 to 500 ng / mL, the concentration of the interleukin-3 is 1 to 500 ng / mL, and the concentration of the interleukin-6 is 1 to 500 ng / mL. A method for producing immune cells differentiated from the stem cells according to

[10] .

[0021]

[12] In step (b''-1), the stem cells are cultured at 35 to 37°C for 7 to 42 days. A method for producing immune cells differentiated from the stem cells according to [9].

[0022]

[13] A kit for producing immune cells differentiated from stem cells, comprising: A coating formation sub-kit used for forming a coating on the surface, A stem cell differentiation sub-kit, and wherein the coating formation sub-kit contains a solvent and a matrix component used for forming a coating solution, the matrix component contains vascular cell adhesion molecule, intercellular adhesion molecule, delta-like ligand 4, and the coating solution is used to be applied to the surface to form the coating on the surface, and the stem cell differentiation sub-kit contains a combination of first cytokines used to be added to a medium to form a medium for stem cell differentiation, the combination of the first cytokines contains stem cell factor, and the combination of the first cytokines contains stem cell factor, and Thrombopoietin, Fms-like tyrosine kinase 3 ligand, Interleukin-7, and a kit comprising the same.

[0023]

[14] In the coating-forming solution, the concentration of the vascular cell adhesion molecule is 0.05 to 50 μg / mL, the concentration of the intercellular adhesion molecule is 0.05 to 50 μg / mL, and the concentration of the δ-like ligand 4 is 0.05 to 50 μg / mL. A kit for producing immune cells differentiated from the stem cells according to

[13] .

[0024]

[15] In the medium for stem cell differentiation, the concentration of the stem cell factor is 1 to 500 ng / mL, the concentration of thrombopoietin is 1 to 500 ng / mL, the concentration of Fms-like tyrosine kinase 3 ligand is 1 to 500 ng / mL, and the concentration of interleukin-7 is 1 to 500 ng / mL. A kit for producing immune cells differentiated from the stem cells according to

[13] .

[0025]

[16] The coating-forming sub-kit further comprises the solvent as a component for forming the matrix component and the coating-forming solution. A kit for producing immune cells differentiated from the stem cells according to

[13] .

[0026]

[17] The stem cell differentiation sub-kit further comprises the medium. A kit for producing immune cells differentiated from the stem cells according to

[13] .

[0027]

[18] Further comprising a stem cell amplification sub-kit, The stem cell amplification sub-kit comprises a combination of second cytokines used to be added into another medium to form a medium for stem cell amplification, The combination of the second cytokines is stem cell factor, thrombopoietin and Fms-like tyrosine kinase 3 ligand, interleukin-3, Interleukin-6 and A kit for producing immune cells differentiated from the stem cells described in

[13] , comprising

[0028]

[19] In the medium for stem cell amplification, the concentration of the stem cell factor is 1 to 500 ng / mL, the concentration of the thrombopoietin is 1 to 500 ng / mL, the concentration of the Fms-like tyrosine kinase 3 ligand is 1 to 500 ng / mL, the concentration of the interleukin-3 is 1 to 500 ng / mL, and the concentration of the interleukin-6 is 1 to 500 ng / mL. A kit for producing immune cells differentiated from the stem cells described in

[18] .

[0029]

[20] The stem cell amplification sub-kit further comprises the other medium. A kit for producing immune cells differentiated from the stem cells described in

[18] .

[0030] In order to make the above and other objects, features and advantages of the present disclosure clearer and easier to understand, preferred embodiments will be given below and described in detail while corresponding to the accompanying drawings.

Brief Description of the Drawings

[0031]

Figure 1

Figure 2A

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Figure 3

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Figure 5A

Figure 5B

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0032] The present disclosure can provide a method for producing immune cells differentiated from stem cells.

[0033] The method for producing immune cells differentiated from stem cells provided by the present disclosure can effectively differentiate stem cells into immune cells, particularly T cells.

[0034] The method for producing immune cells differentiated from the stem cells of the present disclosure described above may include the following steps, but is not limited thereto.

[0035] First, a coating containing a matrix is formed on the surface. The surface may include at least one surface of a cell culture device, but is not limited thereto. Examples of the cell culture device may include, but are not limited to, a cell culture container, a cell culture membrane, and a cell culture scaffold.

[0036] In one embodiment, the cell culture device may be a cell culture container, and the at least one surface may include the inner surface of the cell culture container. In this embodiment, the inner surface may include, but is not limited to, the bottom surface and / or the side wall surface inside the cell culture container. In another embodiment, the cell culture device may be a cell culture membrane, and the at least one surface may include the upper surface and / or the lower surface of the cell culture membrane. In yet another embodiment, the cell culture device may be a cell culture scaffold, and the at least one surface may include the outer surface of the cell culture scaffold and / or the surface inside its pores.

[0037] The matrix may include, but is not limited to, vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and delta-like ligand 4 (DLL4).

[0038] There is no particular limitation on the method of forming the coating on the surface, and any method that can form a coating containing the matrix on the surface may be used. In one embodiment, the coating may be formed from a coating-forming solution, and the coating-forming solution may contain the matrix. There is also no particular limitation on the method of forming the coating with the coating-forming solution, and any method that can finally form a coating containing the matrix on the surface with the coating-forming solution may be used. In one embodiment, the coating-forming solution is brought into contact with the surface so that the coating-forming solution covers the surface, thereby forming a coating containing the matrix on the surface.

[0039] In one embodiment, in the coating-forming solution, the concentration of the vascular cell adhesion molecule in the matrix may be about 0.05 to 50 μg / mL, such as about 0.1 to 50 μg / mL, about 0.5 to 45 μg / mL, about 0.5 to 40 μg / mL, about 0.75 to 35 μg / mL, about 1 to 30 μg / mL, about 2 to 25 μg / mL, about 2.5 to 20 μg / mL, about 4 to 15 μg / mL, about 5 to 10 μg / mL, about 0.1 to 10 μg / mL, about 0.05 μg / mL, about 0.06 μg / mL, about 0.07 μg / mL, about 0.08 μg / mL, about 0.09 μg / mL, about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, about 0.5 μg / mL, about 0.6 μg / mL, about 0.7 μg / mL, about 0.8 μg / mL, about 0.9 μg / mL, about 1 μg / mL, about 2 μg / mL, about 2.5 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 12.5 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, etc., but is not limited thereto. In a specific embodiment, in the coating-forming solution, the concentration of the vascular cell adhesion molecule in the matrix may be about 10 μg / mL. In another specific embodiment, in the coating-forming solution, the concentration of the vascular cell adhesion molecule in the matrix may be about 0.1 μg / mL. In yet another specific embodiment, in the coating-forming solution, the concentration of the vascular cell adhesion molecule in the matrix may be about 1 μg / mL.

[0040] In one embodiment, in the coating-forming solution, the concentration of the intercellular adhesion molecule in the matrix may be about 0.05 to 50 μg / mL, such as about 0.1 to 50 μg / mL, about 0.5 to 45 μg / mL, about 0.5 to 40 μg / mL, about 0.75 to 35 μg / mL, about 1 to 30 μg / mL, about 2 to 25 μg / mL, about 2.5 to 20 μg / mL, about 4 to 15 μg / mL, about 5 to 10 μg / mL, about 0.1 to 10 μg / mL, about 0.05 μg / mL, about 0.06 μg / mL, about 0.07 μg / mL, about 0.08 μg / mL, about 0.09 μg / mL, about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, about 0.5 μg / mL, about 0.6 μg / mL, about 0.7 μg / mL, about 0.8 μg / mL, about 0.9 μg / mL, about 1 μg / mL, about 2 μg / mL, about 2.5 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 12.5 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, etc., but is not limited thereto. In a specific embodiment, in the coating-forming solution, the concentration of the intercellular adhesion molecule in the matrix may be about 10 μg / mL. In another specific embodiment, in the coating-forming solution, the concentration of the intercellular adhesion molecule in the matrix may be about 0.1 μg / mL. In yet another specific embodiment, in the coating-forming solution, the concentration of the intercellular adhesion molecule in the matrix may be about 1 μg / mL.

[0041] In one embodiment, in the coating-forming solution, the concentration of the δ-like ligand in the matrix is about 0.05 to 50 μg / mL, such as about 0.1 to 50 μg / mL, about 0.5 to 45 μg / mL, about 0.5 to 40 μg / mL, about 0.75 to 35 μg / mL, about 1 to 30 μg / mL, about 2 to 25 μg / mL, about 2.5 to 20 μg / mL, about 4 to 15 μg / mL, about 5 to 10 μg / mL, about 0.1 to 10 μg / mL, about 0.05 μg / mL, about 0.06 μg / mL, about 0.07 μg / mL, about 0.08 μg / mL, about 0.09 μg / mL, about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, about 0.5 μg / mL, about 0.6 μg / mL, about 0.7 μg / mL, about 0.8 μg / mL, about 0.9 μg / mL, about 1 μg / mL, about 2 μg / mL, about 2.5 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 12.5 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, etc., but is not limited thereto. In a specific embodiment, in the coating-forming solution, the concentration of the δ-like ligand in the matrix may be about 10 μg / mL. In another specific embodiment, in the coating-forming solution, the concentration of the δ-like ligand in the matrix may be about 0.1 μg / mL. In yet another specific embodiment, in the coating-forming solution, the concentration of the δ-like ligand in the matrix may be about 1 μg / mL.

[0042] In another embodiment, in the coating-forming solution, the concentrations of vascular cell adhesion molecule, intercellular adhesion molecule, and δ-like ligand in the matrix are each independently about 0.05 to 50 μg / mL, such as about 0.1 to 50 μg / mL, about 0.5 to 45 μg / mL, about 0.5 to 40 μg / mL, about 0.75 to 35 μg / mL, about 1 to 30 μg / mL, about 2 to 25 μg / mL, about 2.5 to 20 μg / mL, about 4 to 15 μg / mL, about 5 to 10 μg / mL, about 0.1 to 10 μg / mL, about 0.05 μg / mL, about 0.06 μg / mL, about 0.07 μg / mL, about 0.08 μg / mL, about 0.09 μg / mL, about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, about 0.5 μg / mL, about 0.6 μg / mL, about 0.7 μg / mL, about 0.8 μg / mL, about 0.9 μg / mL, about 1 μg / mL, about 2 μg / mL, about 2.5 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 12.5 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, etc., but are not limited thereto. In a specific embodiment, in the coating-forming solution, the concentrations of vascular cell adhesion molecule, intercellular adhesion molecule, and δ-like ligand in the matrix may all be about 10 μg / mL. In another specific embodiment, in the coating-forming solution, the concentrations of vascular cell adhesion molecule, intercellular adhesion molecule, and δ-like ligand in the matrix may all be about 0.1 μg / mL. In yet another specific embodiment, in the coating-forming solution, the concentrations of vascular cell adhesion molecule, intercellular adhesion molecule, and δ-like ligand in the matrix may all be about 1 μg / mL.

[0043] Furthermore, in one embodiment, in the coating-forming solution or the coating, the content ratio of vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand in the matrix is about 0.01 to 50:0.01 to 50:0.01 to 50, for example, about 0.05 to 50:0.05 to 50:0.05 to 50, about 0.1 to 45:0.1 to 45:0.1 to 45, about 0.5 to 40:0.5 to 40:0.5 to 40, about 1 to 35:1 to 35:1 to 35, about 2 to 30:2 to 30:2 to 30, about 2.5 to 25:2.5 to 25:2.5 to 25, about 4 to 20:4 to 20:4 to 20, about 5 to 15:5 to 15:5 to 15, about 0.1 to 10:0.1 to 10:0.1 to 10, about 1:1:1, about 1:2:1, about 1:1:2, about 1:2:2, about 2:1:1, about 2:2:1, about 1:3:1, about 1:1:3, about 1:3:3, about 3:1:1, about 3:3:1, about 1:4:1, about 1:1:4, about 1:4:4, about 4:1:1, about 4:4:1, about 1:5:1, about 1:1:5, about 1:5:5, about 5:1:1, about 5:5:1, about 1:6:1, about 1:1:6, about 1:6:6, about 6:1:1, about 6:6:1, about 1:7:1, about 1:1:7, about 1:7:7, about 7:1:1, about 7:7:1, about 1:8:1, about 1:1:8, about 1:8:8, about 8:1:1, about 8:8:1, about 1:9:1, about 1:1:9, about 1:9:9, about 9:1:1, about 9:9:1, about 1:10:1, about 1:1:10, about 1:10:10, about 10:1:1, about 10:10:1, about 1:50:1, about 1:1:50, about 1:50:50, about 50:1:1, about 50:50:1, about 1:100:1, about 1:1:100, about 1:100:100, about 100:1:1, about 100:100:1, etc., but is not limited thereto. In a specific embodiment, in the coating-forming solution or the coating, the content ratio of vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand in the matrix may be about 1:1:1.

[0044] Furthermore, in the method for producing immune cells differentiated from the stem cells of the present disclosure, after the step of forming a coating containing a matrix on the surface, the stem cells are cultured on the coating in the presence of a first combination of cytokines, and the stem cells are differentiated into immune cells.

[0045] The source of the stem cells may include, but is not limited to, umbilical cord blood or induced pluripotent stem cells (iPSCs). Further, the stem cells may be CD34 positive, but are not limited thereto. In one embodiment, the stem cells are hematopoietic stem cells (HSCs). Further, the immune cells may include, but are not limited to, gamma delta T cells (GDT cells), NK cells, and the like.

[0046] Also, the combination of the first cytokines may include, but is not limited to, stem cell factor (SCF), thrombopoietin (TPO), Fms-like tyrosine kinase 3 ligand (Flt3L), and interleukin-7 (IL-7).

[0047] In one embodiment, in the above-described step of culturing stem cells on the coating and differentiating the stem cells into immune cells in the presence of a combination of a first cytokine, the concentration of stem cell factor is about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of stem cell factor may be about 100 ng / mL. In another specific embodiment, the concentration of stem cell factor may be about 8 ng / mL. In yet another embodiment, the concentration of stem cell factor may be about 20 ng / mL. In still another embodiment, the concentration of stem cell factor may be about 50 ng / mL.

[0048] Also, in one embodiment, in the above step of culturing the stem cells on the coating in the presence of the combination of the first cytokines to differentiate the stem cells into immune cells, the concentration of thrombopoietin is about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of thrombopoietin may be about 100 ng / mL. In another specific embodiment, the concentration of thrombopoietin may be about 8 ng / mL. In yet another embodiment, the concentration of thrombopoietin may be about 20 ng / mL. In still another embodiment, the concentration of thrombopoietin may be about 50 ng / mL.

[0049] In one embodiment, in the above step of culturing stem cells on the coating in the presence of a combination of the first cytokines to differentiate the stem cells into immune cells, the concentration of Fms-like tyrosine kinase 3 ligand is about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 100 ng / mL. In another specific embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 8 ng / mL. In yet another embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 20 ng / mL. In still another embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 50 ng / mL.

[0050] Also, in one embodiment, in the above step of culturing the stem cells on the coating in the presence of the combination of the first cytokines to differentiate the stem cells into immune cells, the concentration of interleukin-7 is 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of interleukin-7 may be about 100 ng / mL. In another specific embodiment, the concentration of interleukin-7 may be about 8 ng / mL. In yet another embodiment, the concentration of interleukin-7 may be about 20 ng / m. In still another embodiment, the concentration of interleukin-7 may be about 50 ng / mL.

[0051] In another embodiment, in the aforementioned step of culturing the stem cells on the coating in the presence of the combination of the first cytokines to differentiate the stem cells into immune cells, the concentration of stem cell factor, the concentration of thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, and the concentration of interleukin-7 are each independently about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but are not limited thereto. In a particular embodiment, the concentration of stem cell factor, the concentration of thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, and the concentration of interleukin-7 may each be about 100 ng / mL. In another particular embodiment, the concentration of stem cell factor, the concentration of thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, and the concentration of interleukin-7 may each be about 8 ng / mL. In yet another embodiment, the concentration of stem cell factor, the concentration of thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, and the concentration of interleukin-7 may each be about 20 ng / mL.In yet another embodiment, the concentration of stem cell factor, the concentration of thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, and the concentration of interleukin-7 may each be about 50 ng / mL.

[0052] Also, in one embodiment, in the aforementioned step of culturing the stem cells on the coating in the presence of the first cytokine combination to differentiate the stem cells into immune cells, the stem cells can be cultured in a medium, which may include, but is not limited to, Iscove’s modified Dulbecco’s medium (IMDM), improved Iscove’s modified Dulbecco’s medium, Roswell Park Memorial Institute (RPMI) medium, and the like. In a specific embodiment, the medium is improved Iscove’s modified Dulbecco’s medium.

[0053] Furthermore, in the aforementioned step of culturing the stem cells on the coating in the presence of the first cytokine combination to differentiate the stem cells into immune cells, the stem cells can be cultured at about 35-37 °C, such as about 35 °C, about 35.5 °C, about 36 °C, about 36.5 °C, about 37 °C, etc., but is not limited thereto.

[0054] In the aforementioned step of culturing the stem cells on the coating in the presence of the first cytokine combination to differentiate the stem cells into immune cells, the stem cells can be cultured for about 7-42 days, such as about 10-40 days, about 12-36 days, about 15-30 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 14 days, about 18 days, about 20 days, about 21 days, about 22 days, about 25 days, about 28 days, etc., but is not limited thereto.

[0055] In one embodiment, a method for producing immune cells differentiated from the stem cells of the present disclosure may further include, but is not limited to, a step of forming a coating containing the aforementioned matrix on a surface, and a step of culturing the stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells, and before the step of culturing the stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells, a step of screening the stem cells from the source of the stem cells.

[0056] In another embodiment, a method for producing immune cells differentiated from the stem cells of the present disclosure may further include, but is not limited to, a step of forming a coating containing the aforementioned matrix on a surface, and a step of culturing the stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells, and before the step of culturing the stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells, a step of amplifying the stem cells.

[0057] In yet another embodiment, a method for producing immune cells differentiated from the stem cells of the present disclosure may further include, but is not limited to, a step of forming a coating containing the aforementioned matrix on a surface, and a step of culturing the stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells, and before the step of culturing the stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells, a step of screening the stem cells from the source of the stem cells, and after screening the stem cells, a step of amplifying the stem cells.

[0058] The process of amplifying the stem cells may include, but is not limited to, a step of culturing the stem cells in the presence of a second cytokine combination.

[0059] The combination of the second cytokine may include, but is not limited to, stem cell factor, the concentration of thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-3 (IL-3), and interleukin-6 (IL-6).

[0060] In one embodiment, in the process of amplifying the stem cells, the concentration of stem cell factor may be about 1 - 500 ng / mL, such as about 5 - 450 ng / mL, about 10 - 400 ng / mL, about 15 - 350 ng / mL, about 20 - 300 ng / mL, about 25 - 250 ng / mL, about 30 - 200 ng / mL, about 35 - 150 ng / mL, about 40 - 100 ng / mL, about 45 - 95 ng / mL, about 50 - 90 ng / mL, about 55 - 85 ng / mL, about 60 - 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of stem cell factor may be about 100 ng / mL. In another specific embodiment, the concentration of stem cell factor may be about 8 ng / mL. In yet another embodiment, the concentration of stem cell factor may be about 20 ng / mL. In still another embodiment, the concentration of stem cell factor may be about 50 ng / mL.

[0061] In one embodiment, in the process of amplifying the stem cells, the concentration of thrombopoietin is about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of thrombopoietin may be about 100 ng / mL. In another specific embodiment, the concentration of thrombopoietin may be about 8 ng / mL. In yet another embodiment, the concentration of thrombopoietin may be about 20 ng / mL. In still another embodiment, the concentration of thrombopoietin may be about 50 ng / mL.

[0062] Also, in one embodiment, in the process of amplifying the stem cells, the concentration of Fms-like tyrosine kinase 3 ligand is about 1 to 500 ng / mL, for example, about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 100 ng / mL. In another specific embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 8 ng / mL. In yet another embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 20 ng / mL. In still another embodiment, the concentration of Fms-like tyrosine kinase 3 ligand may be about 50 ng / mL.

[0063] Also, in one embodiment, in the process of amplifying the stem cells, the concentration of interleukin-3 is about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of interleukin-3 may be about 100 ng / mL. In another specific embodiment, the concentration of interleukin-3 may be about 8 ng / mL. In yet another embodiment, the concentration of interleukin-3 may be about 20 ng / mL. In still another embodiment, the concentration of interleukin-3 may be about 50 ng / mL.

[0064] Also, in one embodiment, in the process of amplifying the stem cells, the concentration of interleukin-6 is about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but is not limited thereto. In a specific embodiment, the concentration of interleukin-6 may be about 100 ng / mL. In another specific embodiment, the concentration of interleukin-6 may be about 8 ng / mL. In yet another embodiment, the concentration of interleukin-6 may be about 20 ng / mL. In still another embodiment, the concentration of interleukin-6 may be about 50 ng / mL.

[0065] In another embodiment, in the process of amplifying the stem cells, the concentration of stem cell factor, thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, the concentration of interleukin-3, and the concentration of interleukin-6 are each independently about 1 to 500 ng / mL, such as about 5 to 450 ng / mL, about 10 to 400 ng / mL, about 15 to 350 ng / mL, about 20 to 300 ng / mL, about 25 to 250 ng / mL, about 30 to 200 ng / mL, about 35 to 150 ng / mL, about 40 to 100 ng / mL, about 45 to 95 ng / mL, about 50 to 90 ng / mL, about 55 to 85 ng / mL, about 60 to 80 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 200 ng / mL, about 225 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, etc., but are not limited thereto. In a particular embodiment, the concentration of stem cell factor, thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, the concentration of interleukin-3, and the concentration of interleukin-6 may all be about 100 ng / mL. In another particular embodiment, the concentration of stem cell factor, thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, the concentration of interleukin-3, and the concentration of interleukin-6 may all be about 8 ng / mL. In yet another embodiment, the concentration of stem cell factor, thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, the concentration of interleukin-3, and the concentration of interleukin-6 may all be about 20 ng / mL.In yet another embodiment, the concentration of stem cell factor, thrombopoietin, the concentration of Fms-like tyrosine kinase 3 ligand, the concentration of interleukin-3, and the concentration of interleukin-6 may all be about 50 ng / mL.

[0066] Also, in one embodiment, in the process of amplifying the stem cells, the stem cells can be cultured in another medium, which may include, but is not limited to, Iscove's modified Dulbecco's medium, improved Iscove's modified Dulbecco's medium, Roswell Park Memorial Institute medium, etc. In a specific embodiment, the other medium is improved Iscove's modified Dulbecco's medium.

[0067] Furthermore, in the process of amplifying the stem cells, the stem cells can be cultured at about 35-37 °C, such as about 35 °C, about 35.5 °C, about 36 °C, about 36.5 °C, about 37 °C, etc., but is not limited thereto.

[0068] Also, in the process of amplifying the stem cells, the stem cells can be cultured for about 5-7 days, such as about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, etc., but is not limited thereto.

[0069] Furthermore, based on the above, the present disclosure can also provide a kit for producing immune cells differentiated from stem cells.

[0070] By using the kit for producing immune cells differentiated from the stem cells of the present disclosure, the stem cells can be effectively differentiated into immune cells, particularly T cells.

[0071] The source of the stem cells may include, but is not limited to, umbilical cord blood or induced pluripotent stem cells. Furthermore, the stem cells may be CD34 positive, but is not limited thereto. In one embodiment, the stem cells are hematopoietic stem cells. Also, in one embodiment, the immune cells may include, but are not limited to, γδ T cells, NK cells, etc.

[0072] A kit for producing immune cells differentiated from the stem cells of the present disclosure may include, but is not limited to, a coating formation sub-kit used to form a coating on the surface and a stem cell differentiation sub-kit.

[0073] The coating formation sub-kit may include, but is not limited to, a matrix component used to form a solvent and a coating formation solution. The matrix component may include, but is not limited to, vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand 4. Further, the coating formation solution may be used to be applied to the surface to form the coating on the surface. The solvent may include, but is not limited to, phosphate buffered saline (PBS).

[0074] There is no particular limitation on the method of forming a coating with the coating formation solution, as long as the coating formation solution can finally form a coating containing the matrix on the surface. In one embodiment, the coating formation solution is brought into contact with the surface so that the coating formation solution covers the surface, thereby forming a coating containing the matrix on the surface.

[0075] In one embodiment, in the matrix component of the coating-forming sub-kit, or in the coating-forming solution or the coating, the content ratio of vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand is about 0.01-50:0.01-50:0.01-50, for example, about 0.05-50:0.05-50:0.05-50, about 0.1-45:0.1-45:0.1-45, about 0.5-40:0.5-40:0.5-40, about 1-35:1-35:1-35, about 2-30:2-30:2-30, about 2.5-25:2.5-25:2.5-25, about 4-20:4-20:4-20, about 5-15:5-15:5-15, about 0.1-10:0.1-10:0.1-10, about 1:1:1, about 1:2:1, about 1:1:2, about 1:2:2, about 2:1:1, about 2:2:1, about 1:3:1, about 1:1:3, about 1:3:3, about 3:1:1, about 3:3:1, about 1:4:1, about 1:1:4, about 1:4:4, about 4:1:1, about 4:4:1, about 1:5:1, about 1:1:5, about 1:5:5, about 5:1:1, about 5:5:1, about 1:6:1, about 1:1:6, about 1:6:6, about 6:1:1, about 6:6:1, about 1:7:1, about 1:1:7, about 1:7:7, about 7:1:1, about 7:7:1, about 1:8:1, about 1:1:8, about 1:8:8, about 8:1:1, about 8:8:1, about 1:9:1, about 1:1:9, about 1:9:9, about 9:1:1, about 9:9:1, about 1:10:1, about 1:1:10, about 1:10:10, about 10:1:1, about 10:10:1, about 1:50:1, about 1:1:50, about 1:50:50, about 50:1:1, about 50:50:1, about 1:100:1, about 1:1:100, about 1:100:100, about 100:1:1, about 100:100:1, etc., but not limited thereto. In a specific embodiment, in the matrix component of the coating-forming sub-kit, or in the coating-forming solution or the coating, the content ratio of vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand may be about 1:1:1.

[0076] The description of the individual concentrations of vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand in the coating-forming solution can refer to the individual descriptions of the concentrations of vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand in the coating-forming solution in the description of the method for producing immune cells differentiated from the stem cells of the present disclosure above, so it will not be repeated here.

[0077] In one embodiment, vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand 4 in the matrix component of the coating-forming sub-kit can be packaged in the same container. In a specific embodiment of this embodiment, the coating-forming solution can be formed by mixing all the contents in the container packaging vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand 4 with a specified amount of the solvent. Also, in another specific embodiment of this embodiment, a specified amount of the contents is taken out from the container packaging vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand 4 and mixed with a specified amount of the solvent, then the coating-forming solution can be formed.

[0078] In another embodiment, vascular cell adhesion molecule, intercellular adhesion molecule, and delta-like ligand 4 in the matrix component of the coating-forming sub-kit may be packaged in different containers respectively. In a specific embodiment of this embodiment, the coating-forming solution can be formed by mixing all the contents in the container packaging vascular cell adhesion molecule, all the contents in the container packaging intercellular adhesion molecule, and all the contents in the container packaging delta-like ligand 4 with a specified amount of the solvent. Also, in another specific embodiment of this embodiment, a specified amount of the contents is taken out from the container packaging vascular cell adhesion molecule, a specified amount of the contents is taken out from the container packaging intercellular adhesion molecule, and a specified amount of the contents is taken out from the container packaging delta-like ligand 4 and mixed with a specified amount of the solvent, then the coating-forming solution can be formed.

[0079] Also, in one embodiment, the coating-forming sub-kit may further include the solvent for forming the matrix component and the coating-forming solution. The solvent may include, but is not limited to, phosphate buffered saline (PBS).

[0080] In a specific embodiment of this embodiment, the matrix component and the solvent may be packaged in the same container to form the coating-forming solution.

[0081] In another specific embodiment of this embodiment, the matrix component and the solvent are packaged in different containers. In this specific embodiment, when using the kit of the present disclosure, all the contents in the container packaging the solvent can be added to the container packaging the matrix component and mixed with the matrix component to form the coating-forming solution, or all the contents in the container packaging the matrix component can be added to the container packaging the solvent and mixed with the solvent to form the coating-forming solution.

[0082] The stem cell differentiation sub-kit in the kit for producing immune cells differentiated from the stem cells of the present disclosure may include, but is not limited to, a combination of first cytokines added to a medium and used to form a medium for stem cell differentiation. The medium may include, but is not limited to, Iscove's modified Dulbecco's medium, improved Iscove's modified Dulbecco's medium, Roswell Park Memorial Institute medium, etc. In a specific embodiment, the medium is improved Iscove's modified Dulbecco's medium.

[0083] The combination of the first cytokines may include, but is not limited to, stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7.

[0084] In one embodiment, among the combinations of the first cytokines of the stem cell differentiation sub-kit, the content ratios of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 are about 1 to 50:1 to 50:1 to 50:1 to 50, for example, about 1 to 45:1 to 45:1 to 45:1 to 45, about 2 to 40:2 to 40:2 to 40:2 to 40, about 3 to 35:3 to 35:3 to 35:3 to 35, about 4 to 30:4 to 30:4 to 30:4 to 30, about 5 to 25:5 to 25:5 to 25:5 to 25, about 6 to 20:6 to 20:6 to 20:6 to 20, about 7 to 15:7 to 15:7 to 15:7 to 15, about 1:1:1:1, about 1:2:1:1, about 1:1:2:1, about 1:1:1:2, about 2:1:1:1, about 1:2:2:1, about 2:2:1:1, about 1:1:2:2, about 2:1:2:1, about 2:1:1:2, about 1:2:1:2, about 1:2:2:2, about 2:1:2:2, about 2:2:1:2, about 2:2:2:1, about 1:3:1:1, about 1:1:3:1, about 1:1:1:3, about 3:1:1:1, about 1:3:3:1, about 3:3:1:1, about 1:1:3:3, about 3:1:3:1, about 3:1:1:3, about 1:3:1:3, about 1:3:3:3, about 3:1:3:3, about 3:3:1:3, about 3:3:3:1, about 1:4:1:1, about 1:1:4:1, about 1:1:1:4, about 4:1:1:1, about 1:4:4:1, about 4:4:1:1, about 1:1:4:4, about 4:1:4:1, about 4:1:1:4, about 1:4:1:4, about 1:4:4:4, about 4:1:4:4, about 4:4:1:4, about 4:4:4:1, about 1:5:1:1, about 1:1:5:1, about 1:1:1:5, about 5:1:1:1, about 1:5:5:1, about 5:5:1:1, about 1:1:5:5, about 5:1:5:1, about 5:1:1:5, about 1:5:1:5, about 1:5:5:5, about 5:1:5:5, about 5:5:1:5, about 5:5:5:1, about 1:6:1:1, about 1:1:6:1, about 1:1:1:6, about 6:1:1:1, about 1:6:6:1, about 6:6:1:1, about 1:1:6:6, about 6:1:6:1, about 6:1:1:6, about 1:6:1:6, about 1:6:6:6, about 6:1:6:6, about 6:6:1:6, about 6:6:6:1, about 1:7:1:1, about 1:1:7:1, about 1:1:1:7, about 7:1:1:1, about 1:7:7:1, about 7:7:1:1, about 1:1:7:7, about 7:1:7:1, about 7:1:1:7, about 1:7:1:7, about 1:7:7:7,It may be, but is not limited to, for example, about 7:1:7:7, about 7:7:1:7, about 7:7:7:1, about 1:8:1:1, about 1:1:8:1, about 1:1:1:8, about 8:1:1:1, about 1:8:8:1, about 8:8:1:1, about 1:1:8:8, about 8:1:8:1, about 8:1:1:8, about 1:8:1:8, about 1:8:8:8, about 8:1:8:8, about 8:8:1:8, about 8:8:8:1, about 1:9:1:1, about 1:1:9:1, about 1:1:1:9, about 9:1:1:1, about 1:9:9:1, about 9:9:1:1, about 1:1:9:9, about 9:1:9:1, about 9:1:1:9, about 1:9:1:9, about 1:9:9:9, about 9:1:9:9, about 9:9:1:9, about 9:9:9:1, about 1:10:1:1, about 1:1:10:1, about 1:1:1:10, about 10:1:1:1, about 1:10:10:1, about 10:10:1:1, about 1:1:10:10, about 10:1:10:1, about 10:1:1:10, about 1:10:1:10, about 1:10:10:10, about 10:1:10:10, about 10:10:1:10, about 10:10:10:1, about 1:50:1:1, about 1:1:50:1, about 1:1:1:50, about 50:1:1:1, about 1:50:50:1, about 50:50:1:1, about 1:1:50:50, about 50:1:50:1, about 50:1:1:10, about 1:50:1:50, about 1:50:50:50, about 50:1:50:50, about 50:50:1:50, about 50:50:50:1, etc. In a specific embodiment, in the combination of the first cytokine of the stem cell differentiation subset, the content ratio of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 may be about 1:1:1:1.,

[0085] Regarding the description of the individual concentrations of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 in the medium for the differentiation of the above-mentioned stem cells, in the description of the method for producing immune cells differentiated from the stem cells of the present disclosure above, in the step of culturing the stem cells on the coating in the presence of the first cytokine combination to differentiate the stem cells into immune cells, reference can be made to the individual descriptions regarding the concentrations of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7, and thus they will not be repeated here.

[0086] In one embodiment, the stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 in the combination of the first cytokines of the stem cell differentiation sub-kit may be packaged in the same container. In a specific embodiment of this embodiment, by mixing all the contents in the container packaging the stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 with a specified amount of the above-mentioned medium, the medium for the differentiation of the stem cells can be formed. Also, in another specific embodiment of this embodiment, if a specified amount of the contents is taken out from the container of the stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 and mixed with a specified amount of the above-mentioned medium, the medium for the differentiation of the stem cells can be formed.

[0087] In another embodiment, the stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 in the combination of the first cytokines of the stem cell differentiation sub-kit may be packaged in different containers. In a specific embodiment of this embodiment, all the contents in the container packaging the stem cell differentiation, all the contents in the container packaging the thrombopoietin, all the contents in the container packaging the Fms-like tyrosine kinase 3 ligand, and all the contents in the container packaging the interleukin-7 are mixed with a specified amount of the medium to form the medium for the stem cell differentiation. Also, in another specific embodiment of this embodiment, a specified amount of the contents is taken out from the container packaging the stem cell differentiation, a specified amount of the contents is taken out from the container packaging the thrombopoietin, a specified amount of the contents is taken out from the container packaging the Fms-like tyrosine kinase 3 ligand, and a specified amount of the contents is taken out from the container packaging the interleukin-7 and mixed with a specified amount of the medium to form the medium for the stem cell differentiation.

[0088] Also, in one embodiment, the stem cell differentiation sub-kit may further include the medium so as to form the combination of the first cytokines and the medium for the stem cell differentiation. The medium may include, but is not limited to, Iscove's modified Dulbecco's medium, improved Iscove's modified Dulbecco's medium, Roswell Park Memorial Institute medium, and the like. In a specific embodiment, the medium is improved Iscove's modified Dulbecco's medium.

[0089] In a specific embodiment of this embodiment, the combination of the first cytokines and the medium may be packaged in the same container to be the medium for the stem cell differentiation.

[0090] In another specific embodiment of this embodiment, the combination of the first cytokine and the medium are packaged in different containers. In this specific embodiment, when using the kit of the present disclosure, all the contents in the container packaging the combination of the first cytokine can be added into the container packaging the medium, mixed with the medium, and the medium for stem cell differentiation can be formed.

[0091] Also, in one embodiment, the kit for producing immune cells differentiated from the stem cells of the present disclosure may further include, but is not limited to, a stem cell amplification sub-kit used for amplifying stem cells.

[0092] The stem cell amplification sub-kit in the kit for producing immune cells differentiated from the stem cells of the present disclosure may include, but is not limited to, a combination of a second cytokine used to be added into another medium to form a medium for stem cell amplification. The other medium may include, but is not limited to, Iscove's modified Dulbecco's medium, improved Iscove's modified Dulbecco's medium, Roswell Park Memorial Institute medium, etc. In a specific embodiment, the other medium is improved Iscove's modified Dulbecco's medium.

[0093] The combination of the second cytokine may include, but is not limited to, stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-3 and interleukin-6.

[0094] In one embodiment, in the combination of the second cytokine of the stem cell amplification sub-kit, the content ratios of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-3 and interleukin-6 are about 1 to 50:1 to 50:1 to 50:1 to 50:1 to 50, for example, about 1 to 45:1 to 45:1 to 45:1 to 45:1 to 45, about 2 to 40:2 to 40:2 to 40:2 to 40:2 to 40, about 3 to 35:3 to 35:3 to 35:3 to 35:3 to 35, about 4 to 30:4 to 30:4 to 30:4 to 30:4 to 30, about 5 to 25:5 to 25:5 to 25:5 to 25:5 to 25, about 6 to 20:6 to 20:6 to 20:6 to 20:6 to 20, about 7 to 15:7 to 15:7 to 15:7 to 15:7 to 15, about 1:1:1:1:1, about 1:2:1:1:1, about 1:1:2:1:1, about 1:1:1:2:1, about 1:1:1:1:2, about 2:1:1:1:1, about 1:2:2:1:1, about 2:2:1:1:1, about 1:1:2:2:1, about 2:1:2:1:1, about 2:1:1:2:1, about 1:2:1:2:1, about 1:1:1:2:2, about 1:2:2:2:1, about 2:1:2:2:1, about 2:2:1:2:1, about 2:2:2:1:1, about 1:1:2:2:2, about 1:3:1:1:1, about 1:1:3:1:1, about 1:1:1:3:1, about 1:1:1:1:3, about 3:1:1:1:1, about 1:3:3:1:1, about 3:3:1:1:1, about 1:1:3:3:1, about 3:1:3:1:1, about 3:1:1:3:1, about 1:3:1:3:1, about 1:1:1:3:3, about 1:3:3:3:1, about 3:1:3:3:1, aboutApproximately 1:5:1:5:1, approximately 1:1:1:5:5, approximately 1:5:5:5:1, approximately 5:1:5:5:1, approximately 5:5:1:5:1, approximately 5:5:5:1:1, approximately 1:1:5:5:5, approximately 1:6:1:1:1, approximately 1:1:6:1:1, approximately 1:1:1:6:1, approximately 1:1:1:1:6, approximately 6:1:1:1:1, approximately 1:6:6:1:1, approximately 6:6:1:1:1, approximately 1:1:6:6:1, approximately 6:1:6:1:1, approximately 6:1:1:6:1, approximately 1:6:1:6:1, approximately 1:1:1:6:6, approximately 1:6:6:6:1, approximately 6:1:6:6:1, approximately 6:6:1:6:1, approximately 6:6:6:1:1, approximately 1:1:6:6:6, approximately 1:7:1:1:1, approximately 1:1:7:1:1, approximately 1:1:1:7:1, approximately 1:1:1:1:7, approximately 7:1:1:1:1, approximately 1:7:7:1:1, approximately 7:7:1:1:1, approximately 1:1:7:7:1, approximately 7:1:7:1:1, approximately 7:1:1:7:1, approximately 1:7:1:7:1, approximately 1:1:1:7:7, approximately 1:7:7:7:1, approximately 7:1:7:7:1, approximately 7:7:1:7:1, approximately 7:7:7:1:1, approximately 1:1:7:7:7, approximately 1:8:1:1:1, approximately 1:1:8:1:1, approximately 1:1:1:8:1, approximately 1:1:1:1:8, approximately 8:1:1:1:1, approximately 1:8:8:1:1, approximately 8:8:1:1:1, approximately 1:1:8:8:1, approximately 8:1:8:1:1, approximately 8:1:1:8:1, approximately 1:8:1:8:1, approximately 1:1:1:8:8, approximately 1:8:8:8:1, approximately 8:1:8:8:1, approximately 8:8:1:8:1, approximately 8:8:8:1:1, approximately 1:1:8:8:8, approximately 1:9:1:1:1, approximately 1:1:9:1:1, approximately 1:1:1:9:1, approximately 1:1:1:1:9, approximately 9:1:1:1:1, approximately 1:9:9:1:1, approximately 9:9:1:1:1, approximately 1:1:9:9:1, approximately 9:1:9:1:1, approximately 9:1:1:9:1, approximately 1:9:1:9:1, approximately 1:1:1:9:9, approximately 1:9:9:9:1, approximately 9:1:9:9:1, approximately 9:9:1:9:1, approximately 9:9:9:1:1, approximately 1:1:9:9:9, approximately 1:10:1:1:1, approximately 1:1:10:1:1, approximately 1:1:1:10:1, approximately 1:1:1:1:10, approximately 10:1:1:1:1, approximately 1:10:10:1:1, approximately 10:10:1:1:1, approximately 1:1:10:10:1, approximately 10:1:10:1:1, approximately 10:1:1:10:1, approximately 1:10:1:10:1, approximately 1:1:1:10:10, approximately 1:10:10:10:1It may be, but is not limited to, about 10:1:10:10:1, about 10:10:1:10:1, about 10:10:10:1:1, about 1:1:10:10:10, about 1:50:1:1:1, about 1:1:50:1:1, about 1:1:1:50:1, about 1:1:1:1:50, about 50:1:1:1:1, about 1:50:50:1:1, about 50:50:1:1:1, about 1:1:50:50:1, about 50:1:50:1:1, about 50:1:1:50:1, about 1:50:1:50:1, about 1:1:1:50:50, about 1:50:50:50:1, about 50:1:50:50:1, about 50:50:1:50:1, about 50:50:50:1:1, about 1:1:50:50:50, etc. In a specific embodiment, in the combination of the first cytokines of the stem cell differentiation subset, the content ratio of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-3 and interleukin-6 may be about 1:1:1:1:1.,

[0095] For the description of the individual concentrations of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-3 and interleukin-6 in the medium for stem cell amplification, reference can be made to the individual descriptions of the concentrations of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-3 and interleukin-6 in the process of stem cell amplification in the description of the method for producing immune cells differentiated from the stem cells of the present disclosure above, so it will not be repeated here.,

[0096] In one embodiment, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, and interleukin-6 in the combination of the second cytokine of the stem cell amplification sub-kit may be packaged in the same container. In a specific embodiment of this embodiment, by mixing all the contents in the container packaging stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, and interleukin-6 with a specified amount of the medium, the medium for stem cell differentiation can be formed. In another specific embodiment of this embodiment, a specified amount of the contents is taken out from the container packaging stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, and interleukin-6 and mixed with a specified amount of the other medium, then the medium for stem cell amplification can be formed.

[0097] In another embodiment, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, and interleukin-6 in the combination of the first cytokine of the stem cell amplification sub-kit may be packaged in different containers. In a specific embodiment of this embodiment, by mixing all the contents in the container packaging stem cell factor, all the contents in the container packaging Fms-like tyrosine kinase 3 ligand, all the contents in the container packaging interleukin-3, and all the contents in the container packaging interleukin-6 with a specified amount of the medium, the medium for stem cell amplification can be formed. In another specific embodiment of this embodiment, a specified amount of the contents is taken out from the container packaging stem cell factor, a specified amount of the contents is taken out from the container packaging Fms-like tyrosine kinase 3 ligand, a specified amount of the contents is taken out from the container packaging interleukin-3, and a specified amount of the contents is taken out from the container packaging interleukin-6 and mixed with a specified amount of the other medium, then the medium for stem cell amplification can be formed.

[0098] In one embodiment, the stem cell amplification sub-kit may further include the other medium as forming a combination of the second cytokine and a medium for stem cell amplification. The other medium may include, but is not limited to, Iscove's Modified Dulbecco's Medium, Modified Iscove's Modified Dulbecco's Medium, Roswell Park Memorial Institute Medium, and the like. In a specific embodiment, the other medium is Modified Iscove's Modified Dulbecco's Medium.

[0099] In a specific embodiment of this embodiment, the combination of the second cytokine and the other medium can be packaged in the same container to serve as the medium for stem cell amplification.

[0100] Furthermore, in another specific embodiment of this embodiment, the combination of the second cytokine and the other medium may be packaged in different containers. In this specific embodiment, when using the kit of the present disclosure, all the contents in the container packaging the combination of the second cytokine are added to the container packaging the other medium and mixed with the other medium to form the medium for stem cell amplification.

[0101] [Examples]

[0102] Example 1

[0103] Isolation of High-Purity Hematopoietic Stem Cells (HSCs) from Umbilical Cord Blood

[0104] Isolation of hematopoietic stem cells (CD34 positive) from the umbilical cord blood of a donor was performed using the EasySep™ Human Cord Blood CD34 Positive Selection Kit II (manufacturer: STEMCELL Technologies).

[0105] First, the CD34-positive cell pre-enrichment reagent in the kit (RosetteSep® Cord Blood CD34 Pre-Enrichment Cocktail II) was added to the donor's umbilical cord blood to form immunorosettes with red blood cells and non-CD34 cells. After subjecting it to density gradient centrifugation, the interface layer rich in CD34-positive cells was collected. Next, the CD34-binding reagent in the kit (EasySep® Human CD34 Positive Selection Cocktail) and magnetic particles (EasySep® Dextran RapidSpheres®) were added to the collected interface layer to form a mixture. Then, the separation tube containing the mixture was set on a magnetic stand (EasySep® EasyStand®) (manufacturer: STEMCELL Technologies) to adsorb the CD34-positive stem cells bound to the magnetic particles in the mixture in the separation tube, while the non-target cells were directly discharged from the separation tube so that the CD34-positive stem cells remained in the separation tube set on the magnetic stand. The separation tube was removed from the magnetic stand, and the cells therein were collected.

[0106] Regarding the collected cells, the CD34 expression of the cells was analyzed by flow cytometry. The results are as shown in Figure 1.

[0107] As can be seen from Figure 1, the CD34 expression of the cells collected by the above-described separation method reached 95%. That is, according to the above-described separation method, highly pure hematopoietic stem cells can be reliably separated from the donor's umbilical cord blood.

[0108] Example 2

[0109] Amplification of hematopoietic stem cells

[0110] Hematopoietic stem cells were separated from the umbilical cord blood of different donors by the method of Example 1.

[0111] Using StemSpan (trademark registered) SFEM II, a commercially available serum-free medium for culturing and amplifying hematopoietic cells, as the basal medium. This is an improved Iscove's modified Dulbecco's medium (IMDM). Human stem cell factor (SCF), human thrombopoietin (TPO), human Fms-like tyrosine kinase 3 ligand (Flt3L), human interleukin-3 (IL-3), and human interleukin-6 (IL-6) were added to the basal medium so that their final concentrations were all 100 ng / mL, respectively, to form a hematopoietic stem cell amplification medium.

[0112] Hematopoietic stem cells isolated from umbilical cord blood with donor code HUCB0100919M and donor code HUCB012023A were each cultured in the hematopoietic stem cell amplification medium for 6 days, and their amplification multiples were calculated. The results are as shown in Figure 2A.

[0113] As can be seen from Figure 2A, after the culture, the amplification multiples of hematopoietic stem cells isolated from umbilical cord blood with donor code HUCB0100919M and donor code HUCB012023A were 44 ± 0.8 times and 9.4 ± 2.3 times, respectively.

[0114] Also, after the culture, for hematopoietic stem cells isolated from umbilical cord blood with donor code HUCB0100919M and donor code HUCB012023A, the expression of CD45 and CD34 of the cells was analyzed by flow cytometry, respectively. The results are as shown in Figure 2B and Figure 2C, respectively.

[0115] As can be seen from Figure 2B and Figure 2C, after the culture, the expression levels of CD45 and CD34 of hematopoietic stem cells isolated from umbilical cord blood with donor code HUCB0100919M and donor code HUCB012023A were maintained at 98% or more and 79% or more, respectively.

[0116] Example 3

[0117] Differentiation from hematopoietic stem cells to γδ T cells and amplification of γδ T cells

[0118] 1. Formation of coating in culture plates

[0119] Vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and delta-like ligand 4 (DLL4) were added to phosphate buffered saline (PBS) to form a coating formation solution (final concentrations: 10 μg / mL for vascular cell adhesion molecule, 10 μg / mL for intercellular adhesion molecule, and 0.1 μg / mL, 1 μg / mL, or 10 μg / mL for delta-like ligand 4).

[0120] 0.5 mL of the coating formation solution was placed in each well of a 24-well culture plate and allowed to stand at room temperature for 2 hours to form a coating on the bottom of each well of the 24-well culture plate. Then, each well was washed once again with phosphate buffered saline to wash away substances not fixed to the bottom surface of each well, completing the preparation of the coating in the culture plate.

[0121] 2. Preparation of medium for promoting differentiation

[0122] Using StemSpan (trademark registered) SFEM II, a commercially available serum-free medium for culturing and amplifying hematopoietic cells, as the basal medium. This is an improved Iscove's modified Dulbecco's medium (IMDM). Stem cell factor (SCF), thrombopoietin (TPO), human Fms-like tyrosine kinase 3 ligand (Flt3L), and interleukin-7 (IL-7) were added to the basal medium such that their respective final concentrations were all 8 ng / mL, 20 ng / mL, 50 ng / mL, or 100 ng / mL, thereby forming a medium that promotes differentiation.

[0123] 3. Differentiation culture - Differentiation from hematopoietic stem cells to γδ T cells

[0124] 1×10 4 hematopoietic stem cells obtained by the above method were cultured in a 24-well culture plate equipped with the above-mentioned coating (in the coating preparation solution, the concentrations of vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and delta-like ligand 4 (DLL4) were all 10 μg / mL, and the content ratio of vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and delta-like ligand 4 (DLL4) was 1:1:1) using the medium that promotes the above-mentioned differentiation for 14 days. In the above-mentioned culture, 0.5 mL of fresh medium that promotes the above-mentioned differentiation was added on the 4th day, and thereafter, it was replaced with 0.5 mL of fresh medium that promotes the above-mentioned differentiation (the final concentrations of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 were all 100 ng / mL) every 3 days.

[0125] After the culture was completed, the expression of γδ TCR in the obtained cells was analyzed by flow cytometry. The results are as shown in Figure 3.

[0126] As can be seen from FIG. 3, after completion of the culture, the expression level of γδ TCR is much higher than 70%, indicating that the culture method can reliably differentiate hematopoietic stem cells into γδ T cells.

[0127] In addition, the coating was replaced with a coating formed from a coating-forming solution containing delta-like ligand 4 (DLL4) at another concentration (0.1 μg / mL and 1 μg / mL), with the concentrations of both vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) maintained at 10 μg / mL, and other culture conditions were the same. As a result, the situation of hematopoietic cells differentiating into γδ T cells was similar to the above results (not shown).

[0128] In addition, when the final concentrations of stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, and interleukin-7 in the medium that promotes the differentiation were all 8 ng / mL, all 20 ng / mL, and all 50 ng / mL, respectively, and other culture conditions were the same as above, the situation of hematopoietic cells differentiating into γδ T cells was also similar to the above results (not shown).

[0129] Example 4

[0130] Evaluation of the toxic effect of γδ T cells differentiated from hematopoietic stem cells stained with Calcein AM on cancer cells

[0131] In this test, lymphoma cells K562 or breast cancer cells MDA-MB-231 were used as target cells (denoted as T) of γδ T cells differentiated from hematopoietic stem cells.

[0132] After staining the target cells with 1 M Calcein AM for 30 minutes, they were washed three times with PBS. Subsequently, effector cells (i.e., γδ T cells differentiated from hematopoietic stem cells) and target cells were co-cultured at an effector cell to target cell ratio (E:T ratio) of 2:1 or 5:1, and the ability of γδ T cells differentiated from hematopoietic stem cells to exert a toxic effect on cancer cells was analyzed.

[0133] After entering live cells, Calcein AM is hydrolyzed by esterase and binds to calcium ions inside the cells, thereby being able to emit fluorescence. When effector cells exert a toxic effect on target cells, the integrity of the target cell membrane is damaged, and the calcium ions inside the target cells are lost, resulting in a change in fluorescence intensity. Therefore, the ability of effector cells to exert a toxic effect on target cells can be evaluated by the change in fluorescence caused by Calcein AM.

[0134] The intensity of fluorescence emission caused by the rupture of target cells induced by 2% Triton-X 100 was defined as complete toxic effect (100%, value A), and the intensity of autofluorescence emission of target cells was defined as value B. Also, the intensity of fluorescence emission caused by the toxic effect of effector cells on target cells was defined as value C.

[0135] The toxic effect of effector cells on target cells was calculated by the following formula.

[0136] Toxic effect (%) = (C - B) / (A - B)×100

[0137] By the above method, the toxic effects of γδ T cells differentiated from hematopoietic stem cells on lymphoma cells K562 and breast cancer cells MDA-MB-231 were evaluated. The results are shown in Figure 4A and Figure 4B respectively.

[0138] As can be seen from Figure 4A, the toxic effect of γδ T cells differentiated from hematopoietic stem cells on lymphoma cells K562 reached 50% or more. Also, as can be seen from Figure 4B, γδ T cells differentiated from hematopoietic stem cells had a dose-dependent toxic effect on breast cancer cells MDA-MB-231.

[0139] Example 5

[0140] Evaluation of the Toxic Effect of γδ T Cells Differentiated from Hematopoietic Stem Cells on Cancer Cells by Real-Time Cell Analysis (RTCA)

[0141] In this study, breast cancer cells MDA-MB-231 were used as target cells (denoted as T) for γδ T cells differentiated from hematopoietic stem cells.

[0142] In the differentiation culture of the experimental group, hematopoietic stem cells isolated from the umbilical cord blood of donors were cultured for a total of 13 days or 26 days using a culture plate containing the coating used in "3. Differentiation culture - Differentiation from hematopoietic stem cells to γδ T cells" in Example 3 and a medium that promotes differentiation (hereinafter referred to as the medium of the present disclosure) to differentiate hematopoietic stem cells into γδ T cells. In the said culture, 0.5 mL of the fresh medium that promotes the said differentiation was added on the 4th day, and thereafter, it was replaced with 0.5 mL of the fresh medium that promotes the said differentiation every 3 days.

[0143] In the differentiation culture of the control group, other culture conditions were the same as those of the cell culture in the experimental group, except that StemSpan (trademark registered) SFEM II, a commercially available serum-free medium used for culturing and amplifying hematopoietic cells, was used as the cell culture medium.

[0144] Real-time cell analysis is a technology based on impedance and micro-sensor electrodes. It is label-free and can continuously monitor the adhesion, morphology, and cell proliferation status of cells in real time. When cells are inoculated into a special sensing well plate in the RTCA device, changes in resistance caused by cell growth and survival are reflected.

[0145] Effector cells (i.e., γδ T cells differentiated from hematopoietic stem cells, denoted as E) and target cells were co-cultured in the device for real-time cell analysis at a cell ratio of effector cells to targets of 1:1 (E:T ratio) to analyze the ability of γδ T cells differentiated from hematopoietic stem cells to exert a toxic effect on cancer cells.

[0146] In the above method, the toxic effects of γδ T cells differentiated from hematopoietic stem cells cultured for 13 days or 26 days on breast cancer cells MDA-MB-231 were evaluated. The results are as shown in FIGS. 5A and 5B respectively.

[0147] As can be seen from FIGS. 5A and 5B, compared with the case of culturing in a commercially available medium, γδ T cells differentiated from hematopoietic stem cells obtained by performing differentiation culture in the medium of the present disclosure, regardless of whether it was cultured for 13 days or 26 days, both had a more excellent ability of toxic effect on cancer cells.

[0148] Example 6

[0149] Effect of intercellular adhesion molecule (ICAM) during coating in differentiation culture on the efficiency of differentiation from hematopoietic stem cells to γδ T cells

[0150] In this test, the differentiation cultures of the experimental group and the control group were basically the same as the culture method of "3. Differentiation culture - differentiation from hematopoietic stem cells to γδ T cells" in Example 3, and only the medium and coating layer used were different.

[0151] Specifically, in the experimental group, the medium prepared in "2. Preparation of a medium for promoting differentiation" in Example 3 was used (hereinafter referred to as the medium of the present disclosure. It contains stem cell factor (SCF) 100 ng / mL, thrombopoietin (TPO) 100 ng / mL, Fms-like tyrosine kinase 3 ligand (Flt3L) 100 ng / mL, and interleukin-7 (IL-7) 100 ng / mL.), and it was divided into three subgroups, namely the non-coated group, the VDI group, and the VD group. The non-coated group does not use a coating during culturing. The VDI group uses a coating containing vascular cell adhesion molecule (VCAM), delta-like ligand 4 (DLL4), and intercellular adhesion molecule (ICAM) during culturing (in the coating preparation solution, the concentrations of vascular cell adhesion molecule (VCAM), delta-like ligand 4 (DLL4), and intercellular adhesion molecule (ICAM) are all 10 μg / mL, and the content ratio of vascular cell adhesion molecule (VCAM), delta-like ligand 4 (DLL4), and intercellular adhesion molecule (ICAM) is 1:1:1.). The VD group uses a coating containing only vascular cell adhesion molecule (VCAM) and delta-like ligand 4 (DLL4) during culturing (in the coating preparation solution, the concentrations of vascular cell adhesion molecule (VCAM) and delta-like ligand 4 (DLL4) are both 10 μg / mL, and the content ratio of vascular cell adhesion molecule (VCAM) to delta-like ligand 4 (DLL4) is 1:1.).

[0152] In contrast, in the control group, StemSpan (trademark registered) SFEM II, a commercially available serum-free medium used for culturing and amplifying hematopoietic cells (hereinafter referred to as the commercially available medium), was used, and it was similarly divided into three subgroups, which were similarly designated as the non-coated group, the VDI group, and the VD group. The non-coated group did not use coating during culture. The VDI group used a coating containing vascular cell adhesion molecule (VCAM), delta-like ligand 4 (DLL4), and intercellular adhesion molecule (ICAM) during culture (in the coating preparation solution, the concentrations of vascular cell adhesion molecule (VCAM), delta-like ligand 4 (DLL4), and intercellular adhesion molecule (ICAM) were all 10 μg / mL, and the content ratio of vascular cell adhesion molecule (VCAM), delta-like ligand 4 (DLL4), and intercellular adhesion molecule (ICAM) was 1:1:1). The VD group used a coating containing only vascular cell adhesion molecule (VCAM) and delta-like ligand 4 (DLL4) during culture (in the coating preparation solution, the concentrations of vascular cell adhesion molecule (VCAM) and delta-like ligand 4 (DLL4) were both 10 μg / mL, and the content ratio of vascular cell adhesion molecule (VCAM) to delta-like ligand 4 (DLL4) was 1:1).

[0153] The preparation method of each coating is basically the same as the coating preparation method described in "1. Formation of Coating in Culture Plate" of Example 3, and the only difference is whether intercellular adhesion molecule (ICAM) is added or not.

[0154] After the differentiation culture was completed, the cells of each group were analyzed by flow cytometry for the expression of unstained state (negative control group) and CD7, CD34, CD56, and γδ TCR. The results are as shown in Figure 6.

[0155] As can be seen from FIG. 6, when cultured on a coating containing only vascular cell adhesion molecule (VCAM) and delta-like ligand 4 (DLL), hematopoietic stem cells, when cultured on a coating containing vascular cell adhesion molecule (VCAM), delta-like ligand 4 (DLL), and intercellular adhesion molecule (ICAM), had a higher expression of γδ TCR and a relatively low level of CD7 expression, which indicates that intercellular adhesion molecule (ICAM) can differentiate hematopoietic stem cells into more mature γδ T cells from precursor T cells.

[0156] Also, it was found from FIG. 6 that when hematopoietic stem cells were cultured in the medium of the present disclosure compared to when cultured in a commercially available medium, the efficiency of differentiation into γδ T cells was more excellent.

[0157] Example 7

[0158] Effect of delta-like ligand 4 (DLL4) in the coating during differentiation culture on the efficiency of hematopoietic stem cells differentiating into γδ T cells

[0159] The differentiation culture in this test is basically the same as the culture method in "3. Differentiation culture - differentiation of hematopoietic stem cells into γδ T cells" of Example 3, except for the coating used.

[0160] Specifically, there are two experimental groups, group IV and group IVD, in this test. The two experimental groups used the medium prepared in "2. Preparation of a medium for promoting differentiation" of Example 3 (hereinafter referred to as the medium of the present disclosure, containing stem cell factor (SCF) 100 ng / mL, thrombopoietin (TPO) 100 ng / mL, Fms-like tyrosine kinase 3 ligand (Flt3L) 100 ng / mL, and interleukin-7 (IL-7) 100 ng / mL).

[0161] However, Group IV used a coating containing only intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) during culturing (in the coating preparation solution, the concentrations of both intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) were 10 μg / mL, and the content ratio of intercellular adhesion molecule (ICAM) to vascular cell adhesion molecule (VCAM) was 1:1.), while Group IVD used a coating containing intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and delta-like ligand 4 (DLL4) during culturing (in the coating preparation solution, the concentrations of intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and delta-like ligand 4 (DLL4) were all 10 μg / mL, and the content ratio of intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and delta-like ligand 4 (DLL4) was 1:1:1.).

[0162] The preparation method of each coating is basically the same as the coating preparation method described in "1. Formation of coating in culture plate" in Example 3, but the only difference is whether delta-like ligand 4 (DLL4) is added or not.

[0163] After the differentiation culture was completed, the expression of CD34, CD56, and γδ TCR in the cells of each group was analyzed by flow cytometry. The results are as shown in Figure 7.

[0164] As can be seen from Figure 7, compared with the case of culturing with a coating containing only (ICAM) and vascular cell adhesion molecule (VCAM), when hematopoietic stem cells were cultured with a coating containing intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and delta-like ligand 4 (DLL), the expression of γδ TCR was higher, and the expression of CD7 was at a relatively low level, indicating that delta-like ligand 4 (DLL4) can differentiate hematopoietic stem cells into more mature γδ T cells from precursor T cells.

[0165] Although the present invention has been disclosed as above in preferred embodiments, this does not limit the present invention, and those skilled in the art can make some changes and modifications as long as they do not depart from the spirit and scope of the present invention. Therefore, the protection scope of the present invention must be determined based on the definition of the appended claims.

Claims

**Claim 1** A method for producing immune cells differentiated from stem cells, comprising: (a) forming a coating containing a matrix on a surface; (b) culturing stem cells on the coating in the presence of a first cytokine combination to differentiate the stem cells into immune cells; wherein the immune cells include gamma delta T cells (GDT cells), the matrix comprises vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), delta-like ligand 4 (DLL4), and the first cytokine combination comprises stem cell factor (SCF), thrombopoietin (TPO), Fms-like tyrosine kinase 3 ligand (Flt3L), interleukin-7 (IL-7), and the source of the stem cells includes umbilical cord blood or induced pluripotent stem cells (iPSCs), and the stem cells are CD34 positive. A method for producing immune cells differentiated from stem cells. **Claim 2** The method for producing immune cells differentiated from stem cells according to claim 1, wherein the coating is formed from a coating-forming solution, and the coating-forming solution contains the matrix. **Claim 3** The method for producing immune cells differentiated from stem cells according to claim 1, wherein in the coating-forming solution, the concentration of the vascular cell adhesion molecule is 0.05 - 50 μg / mL, the concentration of the intercellular adhesion molecule is 0.05 - 50 μg / mL, and the concentration of the delta-like ligand 4 is 0.05 - 50 μg / mL. **Claim 4** The method for producing immune cells differentiated from stem cells according to claim 1, wherein in step (b), the concentration of the stem cell factor is 1 - 500 ng / mL, the concentration of the thrombopoietin is 1 - 500 ng / mL, the concentration of the Fms-like tyrosine kinase 3 ligand is 1 - 500 ng / mL, and the concentration of the interleukin-7 is 1 - 500 ng / mL.

5. The method for producing immune cells differentiated from stem cells according to claim 1, wherein in step (b), the stem cells are cultured at 35 to 37 ° C for 7 to 42 days.

6. The method for producing immune cells differentiated from stem cells according to claim 1, further comprising, prior to step (b), (b') screening the stem cells from the source of the stem cells.

7. The method for producing immune cells differentiated from stem cells according to claim 1, further comprising, prior to step (b), (b'') amplifying the stem cells.

8. Prior to step (b), (b') screening the stem cells from the source of the stem cells; and (b'') amplifying the stem cells after step (b'); The method for producing immune cells differentiated from stem cells according to claim 1, further comprising.

9. The process of amplifying the stem cells comprises (b''-1) culturing the stem cells in the presence of a second cytokine combination, the method for producing immune cells differentiated from stem cells according to claim 7 or 8.

10. The second cytokine combination is stem cell factor; thrombopoietin; Fms-like tyrosine kinase 3 ligand; interleukin-3 (IL-3); interleukin-6 (IL-6); The method for producing immune cells differentiated from stem cells according to claim 9, comprising.

11. In step (b''), the concentration of the stem cell factor is 1 to 500 ng / mL, the concentration of the thrombopoietin is 1 to 500 ng / mL, the concentration of the Fms-like tyrosine kinase 3 ligand is 1 to 500 ng / mL, the concentration of the interleukin-3 is 1 to 500 ng / mL, and the concentration of the interleukin-6 is 1 to 500 ng / mL. The method for producing immune cells differentiated from stem cells according to claim 10.

12. In step (b''-1), the stem cells are cultured at 35 to 37 ° C for 7 to 42 days. The method for producing immune cells differentiated from stem cells according to claim 9.

13. A kit for producing immune cells differentiated from stem cells, comprising a coating-forming sub-kit used to form a coating on the surface; a stem cell differentiation sub-kit; and The coating-forming sub-kit contains a solvent and a matrix component used to form a coating-forming solution, wherein the matrix component comprises a vascular cell adhesion molecule, an intercellular adhesion molecule, delta-like ligand 4, and the coating-forming solution is applied to the surface and used to form the coating on the surface, and the stem cell differentiation sub-kit contains a combination of first cytokines that are added to a medium and used to form a medium for stem cell differentiation, wherein the combination of the first cytokines comprises stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-7, and is a kit for producing immune cells differentiated from stem cells.

14. In the coating-forming solution, the concentration of the vascular cell adhesion molecule is 0.05 to 50 μg / mL, the concentration of the intercellular adhesion molecule is 0.05 to 50 μg / mL, and the concentration of delta-like ligand 4 is 0.05 to 50 μg / mL. The kit for producing immune cells differentiated from stem cells according to claim 13.

15. In the medium for stem cell differentiation, the concentration of the stem cell factor is 1 to 500 ng / mL, the concentration of the thrombopoietin is 1 to 500 ng / mL, the concentration of the Fms-like tyrosine kinase 3 ligand is 1 to 500 ng / mL, and the concentration of the interleukin-7 is 1 to 500 ng / mL. The kit for producing immune cells differentiated from stem cells according to claim 13.

16. The coating-forming sub-kit further contains the solvent as the one that forms the matrix component and the coating-forming solution. The kit for producing immune cells differentiated from stem cells according to claim 13.

17. The stem cell differentiation sub-kit further contains the medium. The kit for producing immune cells differentiated from stem cells according to claim 13.

18. It further contains a stem cell amplification sub-kit, wherein the stem cell amplification sub-kit contains a combination of second cytokines that are added to another medium and used to form a medium for stem cell amplification, wherein the combination of the second cytokines comprises stem cell factor, thrombopoietin, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and is a kit for producing immune cells differentiated from stem cells according to claim 13.

19. In the medium for stem cell amplification, the concentration of the stem cell factor is 1 to 500 ng / mL, the concentration of the thrombopoietin is 1 to 500 ng / mL, the concentration of the Fms-like tyrosine kinase 3 ligand is 1 to 500 ng / mL, the concentration of interleukin-3 is 1 to 500 ng / mL, and the concentration of interleukin-6 is 1 to 500 ng / mL. A kit for producing immune cells differentiated from the stem cells according to claim 18.

20. The kit for producing immune cells differentiated from the stem cells according to claim 18, wherein the stem cell amplification sub-kit further contains the other medium.

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