Method for producing natural killer cells

Abstract

The present invention provides a method for producing natural killer (NK) cells or precursor cells thereof from pluripotent stem cells, comprising a) a step of in vitro inducing hematopoietic progenitor cells (HPC) from pluripotent stem cells, b) a step of in vitro expanding HPC, and c) a step of in vitro inducing NK cells or precursor cells thereof from HPC and inhibiting histone methyltransferase and / or aromatic hydrocarbon receptors (AHR) in one or more of steps a) to c); and a method comprising (1) a step for forming pluripotent stem cell spheres having an average particle diameter of 200 [mu] m or more in a first culture medium, (2) a step for inducing the pluripotent stem cell spheres obtained in step (1) into a cell population containing HPC by three-dimensional culture using a second culture medium containing a histone methyltransferase inhibitor and / or an AHR antagonist, and (3) a step for inducing the pluripotent stem cell spheres obtained in step (2) into a cell population containing HPC. And (3) a step in which the cell population containing HPC obtained in step (2) is induced into a cell population containing NK cells or precursor cells thereof by three-dimensional culture using a third culture medium containing a histone methyltransferase inhibitor and / or an AHR antagonist, and one or more of steps (1) to (3) are performed by a perfusion culture method. Steps (1) to (3) are preferably performed by a continuous perfusion culture method.

Description

Technical Field

[0001] This invention relates to a method for efficiently and massively producing natural killer cells (hereinafter, sometimes simply referred to as "NK cells") useful in the medical field from pluripotent stem cells. Background Technology

[0002] Numerous methods for inducing NK cells from pluripotent stem cells have been reported. For example, Patent Document 1 describes a method for inducing NK cells from pluripotent stem cell spheroids via hematopoietic progenitor cells (hereinafter, sometimes simply referred to as "HPC"). The inventors have discovered that by increasing the average particle size of the spheroids of pluripotent stem cells formed in three-dimensional culture to a larger size than previously possible, and by combining this with a perfusion culture method, NK cells can be induced efficiently and rapidly, and this has led to a patent application (Patent Document 2).

[0003] On the other hand, research on culture medium additives has yielded various findings. For example, Patent Document 3 discloses a method for inducing NK cells using Notch ligands. Furthermore, Patent Document 4 discloses a method for inducing lymphocytes using Notch ligands and inhibitors of histone methyltransferase EZH1, a component of the multicomb complex; and further reports a method for inducing T precursor cells using Notch ligands and StemRegenin 1 (SR1), an aryl hydrocarbon receptor (AHR) antagonist (Patent Document 5); and a method for inducing NK cells from CD34-positive cells using SR1 and thrombopoietin (TPO) (Patent Document 6).

[0004] However, the NK cell induction effects of histone methyltransferase inhibition or AHR inhibition alone are unclear. In addition, there are no reports on the effects of combining histone methyltransferase inhibition with AHR inhibition.

[0005] Against this backdrop, there is a need for NK cell induction methods that can further improve induction efficiency.

[0006] Existing technical documents Patent documents Patent Document 1: WO 2020 / 086889 Patent Document 2: WO 2023 / 145922 Patent Document 3: US 8,518,397 Patent Document 4: WO 2018 / 048828 Patent Document 5: WO 2021 / 200901 Patent document 6: WO 2017 / 046142. Summary of the Invention

[0007] The problem that the invention aims to solve Therefore, the object of this invention is to provide a method for inducing NK cells from pluripotent stem cells more efficiently than existing technologies. A further object of this invention is to provide a novel method for inducing NK cells from pluripotent stem cells, which, by combining perfusion culture, can further improve the induction efficiency achieved by this method.

[0008] Methods for solving problems To achieve the above-mentioned objectives, the inventors conducted various studies and found that, in the method for in vitro induction of NK cells from pluripotent stem cells via HPC, NK cells can be induced efficiently and rapidly by inhibiting histone methyltransferases and / or AHR in one or more of the following steps: a) Steps for inducing HPC from pluripotent stem cells, b) Steps for amplifying HPC, and c) Steps for inducing NK cells or their precursor cells by HPC.

[0009] Furthermore, by performing steps a) to c) using three-dimensional culture, the average particle size of the pluripotent stem cell spheroids formed in step a) is made to be greater than 200 μm. Cell populations containing HPC are induced from these spheroids, and perfusion culture is combined in at least a portion of the subsequent culture, thereby achieving more efficient and better induction of NK cells, thus completing the present invention.

[0010] That is, the present invention is as follows.

[0011] [Project 1] A method for producing natural killer (NK) cells or their precursor cells from pluripotent stem cells, characterized in that the method comprises: a) The procedure of inducing hematopoietic progenitor cells (HPCs) from pluripotent stem cells in vitro. b) In vitro, the steps for amplifying HPC, and c) The step of inducing NK cells or their precursor cells by HPC in vitro. Furthermore, histone methyltransferases and / or aryl hydrocarbon receptors (AHRs) are inhibited in one or more of steps a) to c).

[0012] [Project 2] According to the method described in Project 1, histone methyltransferase catalyzes the addition of methyl groups to histone 3 lysine residue 27 (H3K27).

[0013] [Project 3] According to the method described in Project 1, the histone methyltransferase is EZH1 (zeste homolog enhancer 1) and / or EZH2 (zeste homolog enhancer 2).

[0014] [Project 4] According to any one of Projects 1 to 3, the inhibition of histone methyltransferase is achieved by a low-molecular-weight inhibitor.

[0015] [Project 5] According to the method described in Project 4, the low molecular weight inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55.

[0016] [Project 6] According to any one of items 1 to 5, the inhibition of AHR is an antagonistic activity against AHR.

[0017] [Project 7] The method according to any one of items 1 to 6, wherein the inhibition of AHR is achieved by a low molecular weight inhibitor.

[0018] [Project 8] According to the method described in Project 7, the low molecular weight inhibitor is SR1 (StemRegenin 1), CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059 or BAY 2416964.

[0019] [Project 9] According to any one of items 1 to 8, in the method, one or more of steps a) to c) are performed by perfusion culture.

[0020] [Project 10] A method for producing NK cells or their precursor cells from pluripotent stem cells, comprising: (1) The step of forming pluripotent stem cell spheroids with an average particle size of more than 200 μm in the first culture medium. (2) The step of inducing the pluripotent stem cell spheroids obtained in step (1) into a cell population containing HPCs through three-dimensional culture in a second medium containing histone methyltransferase inhibitors and / or AHR antagonists, and (3) The step of inducing the cell population containing HPC obtained in step (2) into a cell population containing NK cells or their precursor cells by three-dimensional culture in a third medium containing histone methyltransferase inhibitors and / or AHR antagonists. And more than one of the steps (1) to (3) is carried out by perfusion culture method.

[0021] [Project 11] According to the method described in Project 10, histone methyltransferase catalyzes the addition of methyl groups to H3K27.

[0022] [Project 12] According to the method described in Project 10, the histone methyltransferase is EZH1 and / or EZH2.

[0023] [Project 13] The method according to any one of items 10 to 12, wherein the histone methyltransferase inhibitor is a low molecular weight inhibitor.

[0024] [Project 14] According to the method described in Project 13, the low molecular weight inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55.

[0025] [Project 15] The method according to any one of items 10 to 13, wherein the AHR antagonist is a low molecular weight inhibitor.

[0026] [Project 16] According to the method described in Project 15, the low molecular weight inhibitor is SR1, CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059 or BAY 2416964.

[0027] [Project 17] According to any one of items 10 to 16, steps (1) to (3) are performed without using a three-dimensional culture carrier and an extracellular matrix.

[0028] [Project 18] According to the method described in Project 17, the perfusion culture in step (1) is carried out using a separation membrane with a pore diameter of 15 to 75 μm.

[0029] [Project 19] According to the method described in Project 17 or 18, the perfusion culture in step (2) is carried out using a separation membrane with a pore diameter of 45 to 225 μm.

[0030] [Project 20] According to any one of items 17 to 19, the perfusion culture in step (3) is carried out using a separation membrane with a pore diameter of 0.2 to 10 μm.

[0031] [Project 21] According to any one of items 16 to 20, steps (1) to (3) are carried out by continuous perfusion culture.

[0032] [Project 22] The method according to any one of items 16 to 21, wherein the first culture medium contains a ROCK inhibitor.

[0033] [Project 23] According to any one of items 16 to 22, the method wherein the second culture medium contains VEGF, BMP4 and GSK3β inhibitor.

[0034] [Project 24] According to the method described in Project 23, the second culture medium further contains a ROCK inhibitor or bFGF.

[0035] [Project 25] According to any one of items 16 to 22, the second culture medium contains SCF, a TGFβ / Smad inhibitor, and VEGF.

[0036] [Project 26] According to the method described in Project 25, the second culture medium further contains a ROCK inhibitor or bFGF.

[0037] [Project 27] The method according to any one of items 16 to 22, wherein the second culture medium comprises SCF and Flt3L.

[0038] [Project 28] According to the method described in Project 27, the second culture medium further comprises at least one selected from ROCK inhibitors, IL-3, and IL-7.

[0039] [Project 29] According to any one of items 16 to 22, the three-dimensional culture in step (2) includes: (2-1) The culture step uses a medium containing VEGF, BMP4, and GSK3β inhibitors as the second medium. (2-2) The culture step using a medium containing SCF, TGFβ / Smad inhibitor, and VEGF as the second medium, and (2-3) The culture step uses a medium containing SCF and Flt3L as the second medium.

[0040] [Project 30] The method according to any one of items 16 to 29, wherein the third culture medium contains IL-15 and SCF.

[0041] [Project 31] According to the method described in Project 30, the third culture medium further comprises IL-7 and Flt3L.

[0042] [Project 32] The method according to any one of items 16 to 31 does not require a sorting step for NK cells.

[0043] Invention Effects By inhibiting histone methyltransferase and / or AHR signaling, this invention enables the efficient, one-time production of large quantities of NK cells from pluripotent stem cells. This method, employing a three-dimensional culture incorporating perfusion culture, allows for extremely efficient and rapid production of NK cells. NK cells produced using this method exhibit high cell-damaging activity against various cancer cells, which is maintained even after freezing, thus facilitating large-scale clinical application. Attached Figure Description

[0044] Figure 1 : This is a graph showing the change in NK cell density over days when the hematopoietic progenitor cells obtained in step (2) are induced to differentiate into NK cells by suspension culture under various conditions.

[0045] Figure 2 : This is a graph showing the changes in CD56-positive cells over days when the hematopoietic progenitor cells obtained in step (2) are induced to differentiate into NK cells under various conditions through suspension culture.

[0046] Figure 3: This shows the FACS analysis results of the expression of CD56 and CD14 in the cell populations after 10, 17, 24, 32 and 39 days of differentiation induction when the hematopoietic progenitor cells obtained in step (2) were induced to differentiate into NK cells under various conditions through suspension culture. Detailed Implementation

[0047] This invention provides a method (hereinafter, sometimes referred to as "the method of this invention") for producing NK cells or their precursor cells from pluripotent stem cells, characterized in that the method comprises: a) The procedure for inducing HPC from pluripotent stem cells in vitro. b) In vitro, the steps for amplifying HPC, and c) The step of inducing NK cells or their precursor cells by HPC in vitro. Furthermore, histone methyltransferase and / or AHR are inhibited in one or more steps from a) to c).

[0048] Examples of pluripotent stem cells used in the method of this invention include embryonic stem (ES) cells and artificial pluripotent stem (iPS) cells, without particular limitation. For example, when using iPS cells, there are no particular limitations on the method of manufacturing iPS cells or the source cells. Furthermore, there are no particular limitations on the method of culturing iPS cells; it can be two-dimensional culture or three-dimensional culture. Moreover, cryopreserved iPS cells can also be used.

[0049] The method of the present invention induces NK cells or their precursor cells in vitro from pluripotent stem cells via HPC. Specifically, the method of the present invention includes: a) The steps for inducing HPC from pluripotent stem cells in vitro. b) In vitro, the steps for amplifying HPC, and c) The step of inducing NK cells or their precursor cells by HPC in vitro. In this process, steps a) and b) can use the same or a series of culture media to continuously maintain and expand HPCs induced from differentiation. This situation, including the maintenance and expansion period, can be regarded as a step of inducing HPCs from pluripotent stem cells. Alternatively, the HPCs obtained from step a) can be temporarily stored, for example, by cryopreservation, and the HPC stock solution can be supplied to step b) after a certain period of storage. For example, when steps a) and b) are carried out by three-dimensional culture based on continuous perfusion culture (for example, in the case of the preferred embodiment of the present invention described later), the process of inducing a cell population containing HPCs from pluripotent stem cell spheroids and further purifying and expanding the HPCs can be performed as a step using the same or a series of culture media (step (2) in the preferred embodiment of the present invention).

[0050] Pluripotent stem cells can be differentiated into NK cells via HPC using well-known methods, such as those described in WO 2020 / 086889, Biochem BiophysRes Commun. 515(1): 1-8 (2019), and Methods Mol Biol. 2048: 107-119 (2019).

[0051] For example, step a) can be carried out by culturing pluripotent stem cells in a medium capable of inducing them into HPCs. Examples of such media include, but are not limited to, media containing inhibitors of vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), and glycogen synthase 3β (GSK3β), media containing stem cell factor (SCF) and inhibitors of transforming growth factor β (TGFβ) / Smad, and media containing SCF and Flt3 ligand (Flt3L).

[0052] For example, step b) can be carried out by culturing HPC with a medium capable of sustaining HPC amplification. Examples of such media include, for instance, a medium containing SCF and Flt3 ligand (Flt3L), but are not limited to them.

[0053] For example, step c) can be carried out by culturing HPCs in a medium capable of inducing HPCs into NK cells. Examples of such media include, for instance, media containing IL-15 and SCF, but are not limited to them.

[0054] Examples of suitable basal culture media for the preliminary steps a) to c) include DMEM / F-12, HEPES (Thermo Fisher Scientific), Essential 6 medium (Thermo Fisher Scientific), Stem Pro-34SFM (Thermo Fisher Scientific), AIM-V Medium (Thermo Fisher Scientific), Stemline II (registered trademark) (Sigma-Aldrich), and ALyS505N-0 (Cell Science Research Institute Co., Ltd.). Those skilled in the art can select the preferred basal culture medium according to the appropriate steps.

[0055] The culture period in steps a) to b) is usually 5 to 20 days, preferably about 10 to 18 days.

[0056] The culture period in step c) is usually 20 to 60 days, preferably about 25 to 40 days.

[0057] The method of the present invention is characterized in that, in one or more steps from a) to c), histone methyltransferase and / or AHR are inhibited.

[0058] In the method of the present invention, the histone methyltransferases used as targets are not particularly limited, but are preferably those that catalyze the addition of methyl groups to histone 3 lysine residue 27 (H3K27), and more preferably EZH1 (zeste homolog enhancer 1) and / or EZH2 (zeste homolog enhancer 2).

[0059] Inhibition of histone methyltransferases can be achieved by adding substances with inhibitory activity to the culture medium. Here, "inhibition of histone methyltransferases" refers to the eventual inhibition of the enzyme-catalyzed methyl transfer reaction to histones, and can be based on any method, including both inhibition of enzyme function and inhibition of expression. Examples of substances that inhibit the function of histone methyltransferases include, for example, low-molecular-weight inhibitors, antibodies, aptamers, etc. On the other hand, examples of substances that inhibit the expression of histone methyltransferases include, for example, nucleic acids complementary to the enzyme gene or its transcript (e.g., siRNA, antisense nucleic acids, miRNA, etc.), but are not limited to these.

[0060] In a preferred embodiment, the histone methyltransferase inhibitor is a low-molecular-weight inhibitor. As a low-molecular-weight inhibitor, a known inhibitor can be selected appropriately based on the target histone methyltransferase. For example, as inhibitors of EZH1 and / or EZH2, examples include EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, and GSK126. Alternatively, for example, inhibitors that are expected to inhibit PRC1.1 and thus inhibit the recruitment of PRC2, thereby inhibiting histone methylation, include, for example, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, L55, etc.

[0061] Among them, GSK503, EPZ005687, EI1, Tazemetostat (EPZ-6438), GSK126, etc. are preferred, and GSK126 is more preferred.

[0062] There are no particular restrictions on the concentration of histone methyltransferase inhibitors, provided that they can efficiently and rapidly induce NK cells without adversely affecting the cells. The preferred concentration can be selected based on the type of inhibitor, and can be chosen from, for example, a range of about 10 nM to about 5 μM, preferably about 100 nM to about 2 μM, and more preferably about 500 nM to about 1 μM. One histone methyltransferase inhibitor can be used alone, or two or more can be used in combination.

[0063] While not wishing to be bound by any particular theory, it suggests, for example, that by using inhibitors of histone methyltransferases such as EZH1 and / or EZH2 (e.g., GSK126), the initiation of differentiation into NK cells induced by HPC can be accelerated, and cell proliferation efficiency increases with longer NK cell differentiation induction time.

[0064] Aromatic hydrocarbon receptors (AHRs) are transcriptional regulators that, when bound to aromatic hydrocarbon compounds as ligands, translocate into the nucleus, thereby regulating the expression of target genes. Inhibition of AHRs can be achieved by adding substances with inhibitory activity against the transcription factor to the culture medium. Here, "inhibition of AHRs" can be based on any method, as long as it ultimately inhibits the transcriptional regulation of the target gene regulated by the transcription factor's expression; it can inhibit not only the function of the transcription factor but also its expression. Examples of substances that inhibit AHR function include, for example, low-molecular-weight inhibitors, antibodies, aptamers, and bait nucleic acids. On the other hand, examples of substances that inhibit AHR expression include, for example, nucleic acids complementary to the AHR gene or its transcription product (e.g., siRNA, antisense nucleic acids, miRNA, etc.), but are not limited to these.

[0065] In a preferred embodiment, the AHR inhibitor (antagonist) is a low-molecular-weight inhibitor. As a low-molecular-weight inhibitor, a known inhibitor is suitable. In a preferred embodiment, examples include SR1 (StemRegenin1), CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, and BAY2416964, with SR1, CH-223191, GNF-351, CB7993113, and 6,2',4'-trimethoxyflavone being more preferred, and SR1 being even more preferred.

[0066] There are no particular restrictions on the concentration of AHR inhibitors, provided that they can efficiently and rapidly induce NK cells without adversely affecting the cells. The preferred concentration can be selected based on the type of inhibitor, and can be chosen from, for example, a range of about 100 nM to about 10 μM, preferably about 500 nM to about 5 μM, and more preferably about 1 μM to about 5 μM. AHR inhibitors can be used alone or in combination of two or more.

[0067] While not wishing to be bound by any particular theory, it suggests that inhibitors of AHR (e.g., SR1) could further enhance cell proliferation efficiency in the early stages of HPC-induced differentiation into NK cells.

[0068] This suggests that histone methyltransferase inhibition and AHR inhibition have different sites of action for effectively inducing NK cells, and therefore, synergistic effects can be expected by inhibiting both. Therefore, in the method of this invention, histone methyltransferase inhibitors and AHR inhibitors can be used individually, but combined use is also preferred. When using histone methyltransferase inhibitors and AHR inhibitors in combination, any of the aforementioned combinations of histone methyltransferase inhibitors and AHR inhibitors can be used; the combined use of GSK126 and SR1 is particularly preferred.

[0069] There are no particular limitations on the inhibition of histone methyltransferases and / or AHR, provided that it is performed in at least one of steps a) to c), and in one embodiment, it may be performed in all steps a) to c). For example, as a preferred timing, performing the inhibition in the latter half of step a) to the first half of step c) may sometimes further enhance the induction efficiency of NK cells.

[0070] The method of this invention is used to efficiently and massively produce NK cells useful in the medical field from pluripotent stem cells. Therefore, although this method can be implemented in two-dimensional culture, it is preferred to implement it in three-dimensional culture for ease of industrial-scale scaling. Furthermore, manually changing the culture medium not only carries the risk of affecting the quality of the final product due to human error, but also anticipates increased manufacturing costs due to increased labor hours. Therefore, the method of this invention is preferably performed using a perfusion culture method that does not require manual culture medium replacement.

[0071] Therefore, in a preferred embodiment, the method of the present invention is a method for producing NK cells or their precursor cells from pluripotent stem cells, comprising: (1) The step of forming pluripotent stem cell spheroids with an average particle size of more than 200 μm in the first culture medium. (2) The step of inducing the pluripotent stem cell spheroids obtained in step (1) into a cell population containing HPCs through three-dimensional culture in a second medium containing histone methyltransferase inhibitors and / or AHR antagonists, and (3) The step of inducing the cell population containing HPC obtained in step (2) into a cell population containing NK cells or their precursor cells by three-dimensional culture in a third medium containing histone methyltransferase inhibitors and / or AHR antagonists. And more than one of the steps (1) to (3) is carried out by perfusion culture method.

[0072] In a particularly preferred embodiment, the aforementioned steps (1) to (3) are performed by continuous perfusion culture. In this embodiment, there is no step between steps (2) and (3) of temporarily storing the cell population containing HPC or the HPC purified from the cell population by means of, for example, cryopreservation. Instead, steps (2) and (3) are performed continuously in one culture system by continuous perfusion culture. Therefore, in step (2), the induction of HPC by differentiation of pluripotent stem cells (as described in step a) and the expansion of HPC (as described in step b) are performed as a single step.

[0073] In addition, in the preferred embodiment, the culturing of pluripotent stem cells is carried out in three-dimensional culture. Therefore, before step (2) (the aforementioned step a)), the aforementioned step (1) is carried out as a pre-step for culturing pluripotent stem cells in three dimensions to create spheres of pluripotent stem cells.

[0074] The following sections provide a detailed description of the three-dimensional culture method and the perfusion culture method.

[0075] (Three-dimensional culture method) The three-dimensional culture method in this invention refers to a method of first forming cell spheres (spheroids) and suspending them in a culture medium for culture (in this specification, it is sometimes referred to as "three-dimensional suspension culture method").

[0076] Three-dimensional suspension culture is a cell culture method characterized by the use of a stirrer or shaker to simultaneously unfold suspended cells, spheroids (spheres), or adherent cells in three dimensions on a culture carrier. Therefore, compared to two-dimensional culture where cells are cultured only on the bottom of the culture container, three-dimensional suspension culture maximizes the number of cells per unit space. Furthermore, the use of a stirrer or shaker for three-dimensional mixing ensures a homogenized culture environment. Additionally, by combining three-dimensional culture with various detectors such as pH sensors and dissolved oxygen sensors, efficient and homogeneous culture can be achieved.

[0077] This allows for maximum utilization of the culture space and enables culture in a uniform environment, which is beneficial for scaling up culture for planned commercial production. Furthermore, when culturing suspension cells and spheroids, it is cost-effective because it eliminates the need for specially processed plastic carriers for cell culture and the extracellular matrix required for large numbers of cultured cells. Moreover, compared to two-dimensional culture where cells adhere to a plane, it allows for culture in an environment closer to that of an organism, making it a useful tool in the manufacture of various cell products derived from pluripotent stem cells (e.g., ES cells, iPS cells, etc.) that require induction into differentiation into various cell types.

[0078] (Perfusion culture method) The three-dimensional culture method of this invention, when combined with perfusion culture, can induce NK cells more efficiently.

[0079] Perfusion culture is a type of continuous culture method. It involves supplying a certain amount of fresh culture medium to the culture medium in the culture container while simultaneously removing a certain amount of culture medium, thereby achieving the goal of continuous culture medium replacement, i.e., the supply of new nutrients and the removal of waste.

[0080] By employing perfusion culture, the need for culture medium replacement is eliminated, thus significantly reducing the operational burden. Furthermore, since this method is typically mechanical, environmental changes such as temperature, pH, dissolved oxygen concentration, and dissolved carbon dioxide concentration fluctuations associated with manual culture medium changes are avoided. Therefore, longer-term cultures can be conducted within the same container, resulting in the maintenance of high cell densities within the same container. Additionally, perfusion culture maintains constant concentrations of various growth factors released by cells that are typically removed during regular culture medium replacements, allowing for culture in an environment closely mimicking the circulatory system of a living organism.

[0081] The separation membrane used in perfusion culture preferably has a certain pore diameter and properties (e.g., degree of hydrophobicity) to prevent the target cell population from escaping from the culture system. The pore diameter of the separation membrane can be appropriately selected based on the target cell population. In the NK cell induction step, since various differentiated cells are induced inside and outside the sphere, the selection of this pore diameter usually requires various studies, and its optimization is very difficult.

[0082] The specific pore diameters in each step are described below. As suitable pore diameters for perfusion culture in this invention, for step (1), the pore diameter is 15-75 μm, for step (2), the pore diameter is 45-225 μm, and for step (3), the pore diameter is 0.2-10 μm.

[0083] There are no particular limitations on the raw materials for the separation membrane. It is preferable to use raw materials that do not affect the components in the cells or culture medium. Examples of such raw materials include metal raw materials such as SUS304 and SUS316; natural fibers such as cellulose fibers; or chemical fibers such as polysulfone fibers and polyethersulfone fibers.

[0084] The three-dimensional culture method of the present invention is characterized by perfusion culture of cell populations, such as cell spheroids or cells, present in the culture system in the following steps. The conditions for each perfusion culture are as described in the following description of each step. In addition, the perfusion culture in the present invention can be "continuously performed perfusion culture".

[0085] Here, "continuous perfusion culture" means that only membrane replacement is performed between each step, and the washing step, which is usually performed without the addition of culture medium replacement, is used to supply culture medium for the subsequent step while removing the culture medium from the previous step.

[0086] For example, in step (1), the culture medium is continuously replaced by simultaneously removing the culture medium while supplying the first culture medium (described later). When switching from step (1) to step (2), the first culture medium is continuously replaced by simultaneously supplying the second culture medium (described later). Similarly, in step (2), the culture medium is continuously replaced by simultaneously supplying the second culture medium and removing the culture medium. When switching from step (2) to step (3), the second culture medium is continuously replaced by simultaneously supplying the third culture medium (described later). Furthermore, in step (3), the culture medium is continuously replaced by simultaneously supplying the third culture medium and removing the culture medium.

[0087] (Regarding the steps for forming spheroids of pluripotent stem cells: Step (1)) In a preferred embodiment of the present invention, step (1) is the step of forming pluripotent stem cell spheroids with an average particle size of 200 μm or more in a first culture medium.

[0088] As mentioned above, pluripotent stem cells used in the spheroidization of pluripotent stem cells in this invention can include ES cells and iPS cells, but there are no particular limitations.

[0089] Spheroidization is performed under suspension culture conditions. There are no particular limitations on the suspension culture conditions, as long as the culture medium environment can be homogenized through three-dimensional mixing using a stirrer or shaker. Furthermore, there are no particular limitations on the container used for suspension culture; containers with stirring mechanisms such as rotary or vibrating stirrers can be used. In the spheroidization of pluripotent stem cells of this invention, the seeding density of pluripotent stem cells is 1.0 × 10⁻⁶. 4Cells / mL to 1.0 × 10⁻⁶ 6 The cell / mL range. If the density is higher than this, the spheroids become too large, sometimes affecting subsequent induction efficiency. More preferably, it is 5.0 × 10⁻⁶. 4 Cells / mL to 2.0 × 10⁻⁶ 5 Inoculate cells at a rate of 1 / mL.

[0090] The culture medium used for spheroidizing pluripotent stem cells in step (1) is not particularly limited, as long as it can be used for the maintenance culture of pluripotent stem cells. Examples of such culture media include StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply Co., Ltd.), mTeSR, etc. TM 1. Culture media capable of feeder-free culture, such as those from STEMCELL Technologies Inc. Among these, the preferred culture medium used in the method of the present invention is one containing a ROCK inhibitor within a feeder-free culture medium. Particularly when using iPS cells as pluripotent stem cells, culture media such as StemFit (registered trademark) AK03N (Ajinomoto Healthy Supply Co., Ltd.) are preferred.

[0091] Examples of ROCK inhibitors used in this invention include Y27632 and Thiazovin. Y27632 is commonly used as a ROCK inhibitor in iPS cell culture. In this invention, its concentration is preferably 1–20 μM, and it is preferred to maintain this concentration for 2 days after cell seeding.

[0092] The culture time required for spheroidization is not limited as long as spheroids can be formed. However, the spheroids in this invention are not the type that are forcibly formed by inserting pluripotent stem cells into pits or the like to force them to merge. Instead, they are formed along with cell proliferation, and therefore require a certain amount of time. For example, 2 to 10 days from cell seeding is preferred, and 4 to 7 days is more preferred.

[0093] The above-mentioned spheroidization can be adjusted by stirring conditions, inoculation density conditions, and culture time. The average particle size of the spheroids in step (1) of the method of the present invention is usually above 200 μm, and is specifically suitable for adjustment in the range of 200 to 600 μm. In order to further improve the production efficiency of NK cells, it is preferred to adjust it to 200 to 500 μm, and even more preferably to adjust it to 200 to 400 μm.

[0094] If the average particle size is less than 200 μm, the culture time will be insufficient to obtain a sufficient number of cells, which will also affect step (2), so it is not preferred. In addition, if the average particle size is too large, the supply of sufficient nutrients and oxygen to the center of the sphere will be stagnant, which will cause cell death such as necrosis, so it is not preferred (see, for example, Cells Tissues Organs 196.1(2012): 34-47).

[0095] The spheroidization step (1) is carried out by perfusion culture. In step (1), perfusion culture can be carried out from the time of seeding of pluripotent stem cells, preferably about one day after the initial seeding of pluripotent stem cells, and more preferably two days after the initial seeding. This is because spheroids are formed after 1 to 2 days of culture, and under these conditions the average particle size is greater than 200 μm. If perfusion culture is carried out before the formation of spheroids, it may not be possible to form spheroids satisfactorily.

[0096] Furthermore, in step (1), the pore diameter of the separation membrane used in perfusion culture is preferably 15–75 μm, more preferably 25–45 μm. This is because, by using a separation membrane with a pore diameter not exceeding the desired spherical size, perfusion culture can be performed while retaining a certain number of spherical bodies within the culture container. Therefore, step (1) preferably includes: (1-1) The step of forming pluripotent stem cell spheroids with an average particle size of more than 200 μm in the first culture medium, and (1-2) The step of maintaining the pluripotent stem cell spheroids with an average particle size of 200 μm or more obtained in step (1-1) by perfusion culture. That is, the pore diameter of the separation membrane in the perfusion culture of step (1) (or step (1-2)) is preferably 15 to 75 μm, more preferably 25 to 45 μm.

[0097] The culture medium replacement during perfusion culture is carried out at a constant dilution rate. Preferably, the spheroidization step of pluripotent stem cells is performed at a rate of 0.01 to 0.2 hours. -1 The dilution rate can be adjusted by referring to factors such as cell seeding density, glucose concentration, lactate concentration, glutamine concentration, and glutamate concentration in the culture supernatant. Adjustments to the dilution rate can be made by placing the culture containers, feed flasks, and discharge flasks on a balance or load sensor, or by monitoring the rotation speed of the peristaltic pump.

[0098] In this step (1), temperature, pH, dissolved oxygen, etc. can be used to control the temperature, pH, and dissolved oxygen concentration to any values ​​desired for the formation of pluripotent stem cell spheres.

[0099] The culture temperature is preferably controlled between 35 and 39°C, more preferably between 36 and 38°C. pH is controlled by the inflow of sterile compressed air, sterile carbon dioxide, a pH adjuster, or the dilution rate of the perfusion culture medium, and its controlled value is preferably controlled between 6.8 and 8.0, more preferably between 7.0 and 7.4. Dissolved oxygen concentration is controlled by sterile compressed air, sterile nitrogen, or sterile oxygen, and its value is controlled between 0 and 6.86 mg / L, preferably maintained at a concentration of 2.00 mg / L or higher. It should be noted that the gas used to maintain pH and dissolved oxygen can be blown into the air layer above the culture medium, blown into the culture medium, or replaced via a permeable membrane; there are no limitations.

[0100] The resulting pluripotent stem cell spheres are then used in a subsequent step (2) to induce hematopoietic progenitor cells.

[0101] (Regarding the steps for inducing spheroids of pluripotent stem cells into a population of cells containing HPCs: Step (2)) In a preferred embodiment of the present invention, step (2) involves inducing the spheroids formed in step (1) into a population of HPC-containing cells through three-dimensional culture in a second culture medium containing a histone methyltransferase inhibitor and / or an AHR antagonist. Here, the histone methyltransferase inhibitor contained in the second culture medium is as described above. Additionally, the AHR antagonist contained in the second culture medium is as described above.

[0102] Here, the “cell population containing HPC” induced by this step refers to the cell population obtained by step (2), which is the concept of HPC population induced inside and outside the sphere.

[0103] This cell population includes: HPCs formed within the sphere, HPCs that escape from the sphere after induction, and cells inside and outside the sphere during the induction process towards HPCs.

[0104] Step (2) can also be performed using the same perfusion culture method as step (1). The pore diameter of the separation membrane used in this step is preferably 45–225 μm, more preferably 45–100 μm. The perfusion culture in this step is performed from the beginning of step (2) until the end of step (2). During this period, the “pluripotent stem cell spheroids” are induced toward a “cell population containing HPCs”.

[0105] During step (2), perfusion culture can be performed by using a separation membrane with the above-mentioned pore diameter, in which a cell population containing a certain amount of HPC is stored in a culture container.

[0106] The culture medium replacement accompanying the perfusion culture in step (2) is also carried out at a certain dilution rate, just as described in step (1). Specifically, as in step (1), it is preferably performed at a dilution rate of 0.01 to 0.2 hr. -1 The dilution rate can be adjusted based on factors such as cell seeding density, glucose concentration, lactate concentration, glutamine concentration, and glutamate concentration in the culture supernatant. This dilution rate also applies when the culture medium used in step (1) is replaced with the culture medium used in step (2).

[0107] As the second culture medium in this step (2), there are no particular limitations as long as it contains histone methyltransferase inhibitors and / or AHR antagonists and is capable of inducing pluripotent stem cells into HPC.

[0108] Examples of such culture media include: a culture medium containing vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), and glycogen synthase 3β (GSK3β) inhibitors (hereinafter referred to as culture medium (2-1)); a culture medium containing stem cell factor (SCF) and transforming growth factor β (TGFβ) / Smad inhibitors (hereinafter referred to as culture medium (2-2)); and a culture medium containing SCF and Flt3 ligand (Flt3L) (hereinafter referred to as culture medium (2-3)). These culture medium components can also be appropriately combined or varied to design suitable induction media. In particular, the timing of administration of histone methyltransferase inhibitors and / or AHR antagonists (specifically, in which of culture media (2-1) to (2-3)) can be appropriately designed. Hereinafter, each of culture media (2-1) to (2-3) will be described as an example of such a culture medium.

[0109] The culture medium (2-1) is a medium containing VEGF, BMP4 and GSK3β inhibitors.

[0110] Here, examples of GSK3β inhibitors include CHIR99021 and SB216763, with CHIR99021 being the preferred one.

[0111] The concentration of VEGF is preferably 1–100 ng / mL, more preferably 50–100 ng / mL. The concentration of BMP4 is preferably 1–100 ng / mL, more preferably 50–100 ng / mL. The concentration of the GSK3β inhibitor when using CHIR99021 is preferably 1–10 μM, more preferably 1–5 μM.

[0112] Furthermore, the culture medium (2-1) may contain a ROCK inhibitor or bFGF. When adding a ROCK inhibitor, those described in step (1) are examples of ROCK inhibitors, with Y27632 being preferred. The concentration of the ROCK inhibitor when using Y27632 is preferably 1 to 20 μM, more preferably 1 to 10 μM. In addition, the concentration of bFGF when added is preferably 1 to 100 ng / mL, more preferably 10 to 50 ng / mL.

[0113] There are no particular limitations on the basal medium used as culture medium (2-1), but it is preferred to use media such as DMEM / F-12, HEPES (Thermo Fisher Scientific), Essential 6 medium (Thermo Fisher Scientific), etc.

[0114] The culture medium (2-2) is a medium containing SCF, TGFβ / Smad inhibitor and VEGF.

[0115] Here, SB431542, LY2157299, and LY2109761 are examples of TGFβ / Smad inhibitors, with SB431542 being preferred. The concentration of SCF is preferably 1–100 ng / mL, more preferably 20–50 ng / mL. When using SB431542, the concentration of the TGFβ / Smad inhibitor is preferably 1–10 μM, more preferably 1–5 μM. The concentration of VEGF is preferably 1–100 ng / mL, more preferably 50–100 ng / mL, similar to that of the culture medium (2-1).

[0116] Furthermore, the culture medium (2-2) may contain ROCK inhibitors or bFGF. Specific examples and concentrations used are the same as those described in the culture medium (2-1).

[0117] There are no particular limitations on the basal medium used as culture medium (2-2), and the same ones as those described in culture medium (2-1) can be used.

[0118] The culture medium (2-3) is a culture medium containing SCF and Flt3L. The concentration of SCF is preferably 1 to 100 ng / mL, more preferably 20 to 50 ng / mL. The concentration of Flt3L is preferably 1 to 100 ng / mL, more preferably 20 to 50 ng / mL.

[0119] Furthermore, the culture medium (2-3) may contain at least one selected from ROCK inhibitors, interleukin (IL)-3, and IL-7. Examples of ROCK inhibitors include those mentioned above, with Y27632 being preferred. The concentration of the ROCK inhibitor when adding Y27632 is preferably 1–20 μM. The concentration of IL-3 when adding IL-3 is preferably 1–100 ng / mL. The concentration of IL-7 when adding IL-7 is preferably 1–100 ng / mL.

[0120] There are no particular limitations on the basal medium used as the culture medium (2-3). A medium suitable for HPC induction can be used, preferably a medium for example, Stem Pro-34 SFM (Thermo Fisher Scientific) with L-glutamine or L-alanyl-L-glutamine added at a final concentration of 1-10 mM.

[0121] It should be noted that the above-mentioned culture media (2-1), (2-2), and (2-3) can be used individually, but in step (2), by replacing culture media (2-1) or (2-2) with (2-3) at an appropriate time, HPC can be induced more efficiently. For example, in the first half of step (2), culture is carried out with culture media (2-1) or (2-2), and in the second half, culture is carried out with culture media (2-3), thereby inducing HPC efficiently.

[0122] At this point, the time for the first and second halves of step (2) can be appropriately set. For example, by setting the first half to 2-6 days and the second half to 3-14 days, HPC can be induced efficiently. The replacement of the culture medium (2-1) or (2-2) with the culture medium (2-3) can be carried out by continuous perfusion operation, or the total amount can be replaced with new culture medium. More preferably, it is ideal to carry out the culture medium replacement by continuous perfusion operation.

[0123] Furthermore, by sequentially changing the culture media (2-1) and (2-2) used in the first half of step (2), HPC can be induced efficiently. Specifically, the culture is first performed using culture medium (2-1), followed by culture using culture medium (2-2).

[0124] At this point, the incubation time of each culture medium can be appropriately set. For example, by setting the incubation time of culture medium (2-1) to 1-3 days and the incubation time of culture medium (2-2) to 1-3 days, HPC can be induced efficiently. The replacement of culture medium (2-1) and culture medium (2-2) can be carried out by continuous perfusion operation, or the total amount can be replaced with new culture medium. More preferably, it is ideal to carry out the culture medium replacement by continuous perfusion operation.

[0125] Therefore, the three-dimensional culture in step (2) is preferably a three-dimensional culture that includes, for example, the following steps: (2-1) The culture step using culture medium (2-1) as the second culture medium, (2-2) Culture step using culture medium (2-2) as the second culture medium (2-3) The culture step using culture medium (2-3) as the second culture medium.

[0126] Each step (2-1) to (2-3) should preferably be performed sequentially.

[0127] In step (2), as described in step (1), temperature, pH, dissolved oxygen, etc., can be controlled to any value desired by inducing hematopoietic progenitor cells using temperature sensors, pH sensors, dissolved oxygen sensors, etc.

[0128] The addition of the histone methyltransferase inhibitor and / or AHR antagonist to the culture medium in step (2) can be carried out in step (2) or only in a part of that step. When the inhibitor (antagonist) is added only in a part of step (2), for example, it can be added only in the latter half of the aforementioned step (2). For example, when step (2) is carried out using culture media (2-1), (2-2), and (2-3) as a second culture medium in sequence, the inhibitor (antagonist) can, for example, be added only in the culture steps using culture media (2-2) and (2-3), or can be added only in the culture step using culture media (2-3), and is not limited to being added only in the latter half of the aforementioned step (2). However, from the viewpoint of efficiently and rapidly inducing NK cells, it is preferable to add either the histone methyltransferase inhibitor or the AHR antagonist only in the culture step using culture media (2-3).

[0129] It should be noted that when adding histone methyltransferase inhibitors and / or AHR antagonists to the culture media in step (2), there are no particular restrictions on the choice of inhibitor (antagonist), as long as it can efficiently and rapidly induce NK cells and does not have adverse effects on the cells. A preferred concentration can be selected according to the type of inhibitor. The concentration of the histone methyltransferase inhibitor can be selected from the range of approximately 10 nM to approximately 5 μM, preferably approximately 100 nM to approximately 2 μM, and more preferably approximately 500 nM to approximately 1 μM. The concentration of the AHR antagonist can be selected from the range of approximately 100 nM to approximately 10 μM, preferably approximately 500 nM to approximately 5 μM, and more preferably approximately 1 μM to approximately 5 μM. One histone methyltransferase inhibitor can be used alone, or two or more can be used in combination. Similarly, one AHR inhibitor can be used alone, or two or more can be used in combination.

[0130] In the method of the present invention, from step (1) to step (3), NK cell populations can be obtained consistently without any special purification steps. Therefore, these cell populations obtained by this step (2) can be supplied directly to the subsequent step (3) on a per-cell-population basis, or they can be supplied to step (3) after selecting only HPCs.

[0131] The inventors have confirmed that the target NK cells can be obtained in step (3) regardless of the method used. Therefore, in the method of the present invention, a step of removing cells other than HPC from the "cell population containing HPC" obtained in step (2) can be added, but it is preferable to directly supply the "cell population containing HPC" obtained in step (2) to step (3).

[0132] NK cells are induced by feeding the resulting "inclusion body cell population" into step (3).

[0133] (Regarding the steps for inducing HPC into NK cells: Step (3)) Step (3) in the method of the present invention is a step of inducing the "cell population containing HPC" obtained in step (2) into a "cell population containing NK cells" by three-dimensional culture in a third culture medium containing a histone methyltransferase inhibitor and / or an AHR antagonist. Here, the histone methyltransferase inhibitor contained in the third culture medium is as described above. In addition, the AHR antagonist contained in the third culture medium is as described above.

[0134] Here, the “cell population containing NK cells” induced by this step refers to a cell population containing NK cells obtained through step (3) and cells in the process of NK cell induction.

[0135] NK cells typically do not form spheroids but exist as single cells. Therefore, according to step (3), a single NK cell population proliferates to form a cell population of multiple single NK cells. This cell population contains the majority of NK cells and a subset of cells undergoing NK cell induction.

[0136] Step (3) can also be performed using the same perfusion culture method as steps (1) and (2). The pore diameter of the separation membrane used is preferably 0.1 to 10 μm, more preferably 0.2 to 5 μm. The perfusion culture in this step is performed continuously from the beginning of step (3) until the end of step (3). During this period, NK cells induced by HPC spheres are generated, but the NK cells are not stored in the spheres and are filtered out separately. Therefore, during step (3), perfusion culture can be performed while storing a certain number of NK cells in the culture container by using a separation membrane that does not exceed the cell diameter of the NK cells.

[0137] Furthermore, the culture medium replacement in step (3) is also performed at a certain dilution rate as described in step (1). Specifically, as in step (1), it is preferable to use a dilution rate of 0.01 to 0.2 hr. -1 The dilution rate can be adjusted by referring to the cell seeding density, glucose concentration, lactate concentration, glutamine concentration, glutamate concentration, etc. in the culture supernatant. This dilution rate is also applicable when the culture medium used in step (2) is replaced with the culture medium used in step (3).

[0138] The third culture medium in step (3) is not particularly limited as long as it contains histone methyltransferase inhibitors and / or AHR antagonists and can induce HPCs into NK cells. Examples of such a medium include those containing IL-15 and SCF. The concentration of IL-15 is preferably 1–100 ng / mL, more preferably 20–50 ng / mL. The concentration of SCF is preferably 1–100 ng / mL, more preferably 20–50 ng / mL. This culture medium may further contain one or more components selected from IL-7, Flt3L, ROCK inhibitors, GSK3β inhibitors, and TGFβ receptor (TGFβR) inhibitors. Examples of ROCK inhibitors include Y27632 and Thiazovin, with Y27632 being preferred. Examples of GSK3β inhibitors include CHIR99021 and SB216763, with CHIR99021 being preferred. Examples of TGFβR inhibitors include LY2157299, SB431542, and LY2109761, with LY2157299 being preferred. In a preferred embodiment, the third culture medium further contains IL-7 and Flt3L in addition to IL-15 and SCF.

[0139] The preferred concentration of IL-7 is 1–100 ng / mL, more preferably 20–50 ng / mL. The preferred concentration of Flt3L is 1–100 ng / mL, more preferably 20–50 ng / mL. The preferred concentration of the ROCK inhibitor is 1–20 μM, more preferably 1–10 μM. The preferred concentration of the GSK3β inhibitor is 1–10 nM, more preferably 1–5 μM. The preferred concentration of the TGFβR inhibitor is 0.1–100 μM, more preferably 0.1–5 μM.

[0140] The culture time in step (3) is usually 20 to 60 days, preferably about 25 to 40 days.

[0141] Furthermore, in step (3), when adding IL-7, Flt3L, ROCK inhibitor, GSK3β inhibitor, or TGFβR inhibitor, the induction efficiency can be improved by adjusting the timing of the addition of these factors. For example, for IL-7 and Flt3L, it is preferable to remove them after day 10 of culture in step (3), and more preferably after day 20. For ROCK inhibitor, GSK3β inhibitor, and TGFβR inhibitor, it is preferable to add them 4 to 7 days before the end of culture.

[0142] The basal culture medium used in step (3) is not particularly limited, and can be, for example, AIM-VMedium (Thermo Fisher Scientific), Stemline II (Sigma-Aldrich), ALyS505N-0 (Cell Science Research Institute, Co., Ltd.), and Stem Pro-34 SFM (Thermo Fisher Scientific), with AIM-V Medium being preferred. Furthermore, it may also contain human serum, fetal bovine serum (FBS), or serum substitutes. Their concentrations are preferably 1–20%, more preferably 1–10%.

[0143] In this step (3), it is further preferred to perform the sphere removal step. The timing of removal can be, for example, before the start of this step, during the implementation of this step, or at the end of this step, but there is no particular limitation and it can be performed at any time.

[0144] The spheres are preferably removed using a cell filter with a mesh size of 20–100 μm, and more preferably using a cell filter with a mesh size of 20–40 μm.

[0145] In addition, in step (3), as described in step (1), temperature, pH, dissolved oxygen, etc. can be used to control the temperature, pH, and dissolved oxygen concentration to any values ​​desired to induce NK cells.

[0146] The addition of the histone methyltransferase inhibitor and / or AHR antagonist to the culture medium in step (3) can be carried out in step (3) or only in a portion of that step. When the inhibitor (antagonist) is added only in a portion of step (3), for example, it can be added only in the first half of step (3) (e.g., until the moment when IL-7 and Flt3L are removed from the culture medium). When the first half of step (3) is carried out as a third culture medium containing IL-7 and Flt3L in addition to IL-15 and SCF, for example, the inhibitor (antagonist) can be added to the culture medium until the moment when IL-7 and Flt3L are removed from the culture medium, but is not limited to this.

[0147] Furthermore, regarding the addition of histone methyltransferase inhibitors and / or AHR antagonists in step (3), there are no particular restrictions on the selection of inhibitors (antagonists), as long as they can efficiently and rapidly induce NK cells without adversely affecting the cells, and the preferred concentration can be appropriately selected according to the type of inhibitor. The concentration of the histone methyltransferase inhibitor can be selected from a range of approximately 10 nM to approximately 5 μM, preferably approximately 100 nM to approximately 2 μM, and more preferably approximately 500 nM to approximately 1 μM. The concentration of the AHR antagonist can be selected from a range of approximately 100 nM to approximately 10 μM, preferably approximately 500 nM to approximately 5 μM, and more preferably approximately 1 μM to approximately 5 μM. One histone methyltransferase inhibitor can be used alone, or two or more can be used in combination. Similarly, one AHR inhibitor can be used alone, or two or more can be used in combination.

[0148] Furthermore, in the method of the present invention, after step (3), a further step of amplification culture of the obtained NK cells or a step of maturation of the NK cells as step (4) can be performed. The amplification culture step or the maturation step can be performed using known methods.

[0149] The method of the present invention is characterized in that steps (1) to (3) or all of steps (1) to (4) are performed without using a three-dimensional culture carrier and an extracellular matrix.

[0150] The method of this invention may further include a step of cryopreserving the obtained NK cells. The NK cells of this invention can be cryopreserved and preserved using known methods after the NK cell manufacturing step.

[0151] The following examples illustrate the invention in more detail, but they are merely illustrative and the invention is not limited thereto. Example

[0152] Example 1: Induction of NK cells from pluripotent stem cell differentiation In this embodiment, human iPS cell lines were used as pluripotent stem cells, and differentiation from iPS cell spheroids to NK cells was achieved through a three-dimensional culture method. After inducing the spheroids of pluripotent stem cells into a cell population containing HPCs, GSK126 or SR1 was added to the culture medium and compared with that without addition. The perfusion culture procedure involved mixing the culture medium using a stirrer, and using pH, temperature, and dissolved oxygen sensors as sensors to continuously monitor and control various management parameters while mixing the culture medium. Furthermore, the perfusion culture was managed using a dedicated pump and balance to control the inflow and outflow rates per unit time.

[0153] Step (1): The steps to form pluripotent stem cell spheroids Pluripotent stem cell spheroidization was achieved using iPS cells cultured in a two-dimensional scale-up environment, reaching a size of 0.25 × 10⁻⁶ cells in a three-dimensional culture vessel. 5 Cells are seeded at a density of cells / mL. Further details are provided below.

[0154] As the culture medium in step (1), a culture medium prepared by adding the ROCK inhibitor Y27632 to StemFit (registered trademark) AK03N (Ajinomoto HealthySupply Co., Ltd.) at a concentration of 10 μM was used.

[0155] In the spheroidization step of pluripotent stem cells, disposable bottles with a total capacity of 500 mL are used as culture containers, and the culture medium is continuously mixed using a stirrer inside the container. The pH is continuously monitored by a pH sensor and controlled to 7.00-7.40 by supplying sterile culture medium, or sterile air, or sterile carbon dioxide.

[0156] Dissolved oxygen concentration was continuously monitored using a dissolved oxygen sensor and maintained above 1.50 mg / L by supplying sterile air or sterile oxygen. The culture temperature was continuously monitored using a temperature sensor and maintained at 37.0°C by externally heating the container with a heater. The culture medium volume was adjusted to 250 mL, and fresh medium was supplied using a pump and balance. The culture medium was drained at the same rate from day 3 to day 5 at 0.04 hr intervals. -1 From day 5 to day 10 of culture, at a rate of 0.06 hr -1The dilution rate was controlled. A SUS316 membrane with a pore diameter of 25 μm was used for separation.

[0157] On day 10 of culture, a cell suspension containing spheroids was collected from the culture medium, and the average particle size and density of the spheroids were determined using a microscope.

[0158] Step (2): The step of inducing spheroids of pluripotent stem cells into a cell population containing hematopoietic progenitor cells. Hematopoietic progenitor cells were prepared by seeding iPS cell spheroids, which were made in step (1), in a three-dimensional culture vessel at a density of 30 spheroids / mL. Other details are as follows.

[0159] As the culture medium used, until day 2, the baseline conditions (condition 1) were the DMEM / F-12, HEPES (Thermo Fisher Scientific) medium supplemented with CHIR99021 to 2 μM, BMP4 to 80 ng / mL, VEGF165 to 80 ng / mL, bFGF to 50 ng / mL, and ROCK inhibitor Y27632 to 10 μM. The experimental conditions were the medium supplemented with SR1 to 2 μM (condition 2) or GSK126 to 1 μM (condition 3). From day 2 to day 4, the baseline conditions (condition 1) were as follows: Essential 6 (Thermo Fisher Scientific) medium supplemented with SCF to 50 ng / mL, VEGF 165 to 80 ng / mL, SB431542 to 2 μM, bFGF to 50 ng / mL, and the ROCK inhibitor Y27632 to 10 μM. Experimental conditions were also performed using medium supplemented with SR1 to 2 μM (condition 2) or GSK126 to 1 μM (condition 3). Furthermore, from day 4 to day 14, the baseline conditions (condition 1) were as follows: the standard hematopoietic progenitor cell differentiation induction basal medium supplemented with SCF to 50 ng / mL and Flt3L to 50 ng / mL. Experimental conditions were also performed using medium supplemented with SR1 to 2 μM (condition 2) or GSK126 to 1 μM (condition 3).

[0160] This step uses disposable bottles with a total capacity of 500 mL as culture containers. The culture medium is continuously mixed using a stirrer inside the container. pH is continuously monitored using a pH sensor and controlled at 7.07 by supplying sterile culture medium, sterile air, or sterile carbon dioxide. Dissolved oxygen concentration is continuously monitored using a dissolved oxygen sensor and controlled to be maintained above 1.50 mg / L by supplying sterile air or sterile oxygen. Culture temperature is continuously monitored using a temperature sensor and controlled at 37.0°C by heating the container externally with a heater. The culture medium volume is adjusted to 250 mL, and new culture medium is supplied using a pump and balance. The culture medium is drained at the same rate from day 1 to day 2 at 0.07 hr intervals. -1 From day 2 to day 4 of culture, at a rate of 0.06 hr -1 From day 5 to day 7 of culture, at 0.13 hr -1 From day 7 to day 12 of culture, at a rate of 0.07 hr -1 From day 12 to day 14 of culture, at a dose of 0.02 hr -1 The dilution rate was controlled. A SUS316 membrane with a pore diameter of 75 μm was used as the separation membrane.

[0161] On day 14 of culture, a suspension of cells with the spheroids removed was collected from the culture medium, and cell surface markers known to be expressed in hematopoietic progenitor cells (CD34, CD43, CD45, CD117) were identified by flow cytometry.

[0162] In the manufacturing process with the addition of GSK126 and SR1, the manufacturing efficiency of hematopoietic progenitor cells was compared with that without the addition, and the expression of the confirmed biomarkers showed no significant difference.

[0163] Step (3): The steps to induce hematopoietic progenitor cells into NK cells NK cell preparation is performed by seeding 25% of the hematopoietic progenitor cell culture medium (the culture medium prepared in step 2 on day 14) containing spheroids in an internal three-dimensional culture vessel. Other details are as follows.

[0164] The basal conditions (Condition 1) were prepared by adding 5% FBS to AIM V Serum Free Medium (Thermo Fisher Scientific), followed by the addition of IL-15, IL-7, SCF, and Flt3L to a final concentration of 50 ng / mL. Experimental conditions were prepared by adding SR1 to 2 μM (Condition 2) or GSK126 to 1 μM (Condition 3).

[0165] All culture containers were 30 mL rotary stirred containers, and suspension culture was carried out in a 5% CO2 incubator at 37°C with a rotation speed of 55 rpm.

[0166] 100 μL of culture medium was collected during culture, and the density of suspended cells was measured using an NC-202 (ChemoMetec) analyzer. The proliferation rate from day 4 of culture was calculated. Figure 1 In conditions 2 and 3 (with the addition of SR1 and GSK126 respectively), the results showed that the cells could be cultured at a higher fold increase compared to the control group in terms of post-passage cell density.

[0167] The NK cell culture medium produced by the method of the present invention was periodically recovered, and the expression of CD56, a cell surface marker known to be expressed in NK cells, was confirmed by flow cytometry. As for the confirmation of target extracellular cells, the expression of CD14, known to be expressed in single cells and macrophages, was confirmed by flow cytometry. Figure 2 and Figure 3 As a result, CD56 expression increased more rapidly in condition 3 (with the addition of GSK126) compared to condition 1, indicating that NK cells could be produced efficiently.

[0168] according to Figure 1 In the early stages of NK cell differentiation induction, SR1 conferred higher cell proliferation efficiency compared to basal conditions. Conversely, in the later stages of differentiation induction, GSK126 conferred high cell proliferation efficiency and, compared to basal conditions, initiated differentiation into CD56-positive cells (NK cells or their precursor cells) more rapidly. Therefore, the combined use of SR1 and GSK126 results in synergistic or complementary effects, predicting a rapid initiation of NK cell differentiation and an overall increase in the proliferation efficiency of the differentiation induction process.

[0169] Industrial practicality The method for manufacturing NK cells derived from pluripotent stem cells of the present invention can be considered a useful method for the mass production of NK cells that can be used in cancer immunotherapy.

[0170] This application is based on Japanese Patent Application No. 2023-126651 filed in Japan on August 2, 2023, the contents of which are incorporated herein by reference in their entirety. Claims (as amended under Article 19 of the Treaty) 1. A method for producing natural killer (NK) cells or their precursor cells from pluripotent stem cells, characterized in that the method comprises: a) The procedure of inducing hematopoietic progenitor cells (HPCs) from pluripotent stem cells in vitro. b) In vitro, the steps for amplifying HPC, and In vitro, the steps for inducing NK cells or their precursor cells by HPC. And in one or more steps from a) to c), histone methyltransferases and / or aryl hydrocarbon receptors (AHRs) are inhibited, and One or more steps from a) to c) are performed by perfusion culture. 2. The method according to claim 1, wherein histone methyltransferase catalyzes the addition of methyl groups to histone 3 lysine residue 27 (H3K27). 3. The method according to claim 1, wherein the histone methyltransferase is EZH1 (zeste homolog enhancer 1) and / or EZH2 (zeste homolog enhancer 2). 4. The method according to claim 1, wherein the inhibition of histone methyltransferase is achieved by a low-molecular-weight inhibitor. 5. The method according to claim 4, wherein the low molecular weight inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55. 6. The method according to claim 1, wherein the inhibition of AHR is an antagonist activity against AHR. 7. The method of claim 1, wherein the inhibition of AHR is achieved by a low-molecular-weight inhibitor. 8. The method according to claim 7, wherein the low molecular weight inhibitor is SR1 (StemRegenin 1), CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059 or BAY2416964. 9. [Deleted]. 10. A method for producing NK cells or their precursor cells from pluripotent stem cells, comprising: (1) The step of forming pluripotent stem cell spheroids with an average particle size of more than 200 μm in the first culture medium. (2) The step of inducing the pluripotent stem cell spheroids obtained in step (1) into a cell population containing HPCs through three-dimensional culture in a second medium containing histone methyltransferase inhibitors and / or AHR antagonists, and (3) The step of inducing the cell population containing HPC obtained in step (2) into a cell population containing NK cells or their precursor cells by three-dimensional culture in a third medium containing histone methyltransferase inhibitors and / or AHR antagonists. And more than one of the steps (1) to (3) is carried out by perfusion culture method. 11. The method of claim 10, wherein histone methyltransferase catalyzes the addition of methyl groups to H3K27. 12. The method according to claim 10, wherein the histone methyltransferase is EZH1 and / or EZH2. 13. The method according to claim 10, wherein the histone methyltransferase inhibitor is a low molecular weight inhibitor. 14. The method according to claim 13, wherein the low molecular weight inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EI1, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55. 15. The method of claim 10, wherein the AHR antagonist is a low molecular weight inhibitor. 16. The method according to claim 15, wherein the low molecular weight inhibitor is SR1, CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059 or BAY 2416964. Explanation or declaration (as amended in accordance with Article 19 of the Treaty) Instructions based on Article 19(1) of the Treaty The applicant amended claim 1 and deleted claim 9. The statement in claim 1 that "one or more steps from a) to c) are performed by perfusion culture" is based on the content of the original claim 9. Furthermore, along with the above modifications, claim 9 of the original claim is deleted. The modified invention is not the invention described in document 1 listed in the international search report, nor is it an invention that would be easily conceived by a person skilled in the art based on documents 1-6.

Claims

1. A method for manufacturing natural killer (NK) cells or precursor cells thereof from pluripotent stem cells, characterized in that, The method comprises: a) a step of inducing hematopoietic progenitor cells (HPCs) from pluripotent stem cells in vitro, b) a step of expanding the HPCs in vitro, and c) a step of inducing NK cells or precursor cells thereof from the HPCs in vitro, and the inhibition of the histone methyltransferase and / or the aryl hydrocarbon receptor (AHR) is performed in one or more of the steps a) to c).

2. The method of claim 1, wherein, The histone methyltransferase catalyzes the addition of a methyl group to histone 3 lysine residue 27 (H3K27).

3. The method of claim 1, wherein, The histone methyltransferase is EZH1 (enhancer of zeste homolog 1) and / or EZH2 (enhancer of zeste homolog 2).

4. The method of claim 1, wherein, The inhibition of the histone methyltransferase is achieved by a low-molecular inhibitor.

5. The method of claim 4, wherein, The low-molecular inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EII, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55.

6. The method according to claim 1, the inhibition of the AHR is with an antagonist activity against the AHR.

7. The method of claim 1, wherein, The inhibition of the AHR is achieved by a low-molecular inhibitor.

8. The method of claim 7, wherein, The low-molecular inhibitor is SR1 (StemRegenin 1), CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, or BAY2416964.

9. The method according to any one of claims 1 to 8, wherein, One or more of the steps a) to c) is performed by perfusion culture.

10. A method for manufacturing NK cells or precursor cells thereof from pluripotent stem cells, comprising: (1) a step of forming pluripotent stem cell spheroids having an average particle size of 200 pm or more in a first culture medium, (2) a step of inducing the pluripotent stem cell spheroids obtained in step (1) into a cell population comprising HPCs by three-dimensional culture using a second culture medium containing a histone methyltransferase inhibitor and / or an AHR antagonist, and (3) a step of inducing the cell population comprising HPCs obtained in step (2) into a cell population comprising NK cells or precursor cells thereof by three-dimensional culture using a third culture medium containing a histone methyltransferase inhibitor and / or an AHR antagonist, and one or more of the steps (1) to (3) is performed by perfusion culture.

11. The method of claim 10, wherein, The histone methyltransferase catalyzes the addition of a methyl group to H3K27.

12. The method of claim 10, wherein, The histone methyltransferase is EZH1 and / or EZH2.

13. The method of claim 10, wherein, The histone methyltransferase inhibitor is a low-molecular inhibitor. The low-molecular inhibitor is GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EII, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55.

14. The method of claim 13, wherein, Low-molecular inhibitors are GSK126, EPZ005687, GSK343, Tazemetostat (EPZ-6438), UNC1999, EBI-2511, PF-06726304, Lirametostat (CPI-1205), EPZ011989, CPI-169, CPI-360, GSK503, EII, OR-S0, OR-S1, DS-3201, FT671, XL177A, P5091, HBX19818, Parthenolide, GNE-6640, XL188, or L55.

15. The method of claim 10, wherein, AHR antagonists are low-molecular inhibitors.

16. The method of claim 15, wherein, Low-molecular inhibitors are SR1, CH-223191, GNF-351, 7-ketocholesterol, CB7993113, 6,2',4'-trimethoxyflavone, PD98059, or BAY 2416964.

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