NK cell culture system and method, and use thereof

Abstract

The present invention relates to the field of immune cell therapy, and specifically relates to a method for expanding NK cells and use thereof. The method comprises using OK-432 in combination with irradiated feeder cells, such as K562 cells, to expand and culture NK cells in vitro.

Description

An NK cell culture system, method and its application Technical Field

[0001] This invention relates to the field of immunotherapy, specifically to a method for expanding NK cells and its application. Background Technology

[0002] NK (Natural Killer Cells) are important immune cells in the body, playing a crucial role in anti-tumor and anti-viral infection immunity. Because the killing activity of NK cells is not MHC-restricted, they are called natural killer cells. The target cells of NK cells are mainly tumor cells, virus-infected cells, certain autologous tissue cells (such as blood cells), and parasites. Therefore, NK cells are an important component of the body's anti-tumor and anti-infection immunity. Due to these characteristics, NK cells have broad application prospects in cell-based immunotherapy. However, the proportion of NK cells in human peripheral blood is very low (approximately 5%-20% of PBMCs), isolating NK cells from peripheral blood is expensive, and the isolated NK cells are generally in an inactive state, which greatly limits their clinical application. Summary of the Invention

[0003] In view of this, one aspect of the present invention provides an NK cell culture system comprising feeder cells, NK cell culture medium I, and NK cell culture medium II; wherein the feeder cells have lost or had their proliferative capacity reduced by irradiation. NK cell culture medium I comprises basal medium, OK-432, and cytokines; NK cell culture medium II comprises basal medium and cytokines.

[0004] In some embodiments, the cell irradiation dose is 100 Gy.

[0005] In some embodiments of the present invention, the trophoblast cells are selected from: K562 cells (human chronic myeloid leukemia cells), lymphoblasts transformed with EBV (Epstein-Barr virus, "EBV"), irradiated PBMCs, and Wilms tumor cells (HFWT).

[0006] In some embodiments, the trophoblast cells are K562 cells.

[0007] In one exemplary embodiment, the K562 cells overexpress the 4-1BBL gene, the IL-15 gene, and the IL-21 gene (referred to as K562-ACP).

[0008] In some embodiments, the final concentration of OK-432 is 0-10 μg / mL.

[0009] In one exemplary embodiment, the final concentration of OK-432 is 1 μg / mL.

[0010] In some embodiments of the above-mentioned NK cell culture system, the initial addition amount of the feeder cells is: a. a concentration of 0-2 × 10⁻⁶. 6 a. cells / mL; or b. the ratio of cells to cultured cells or cell populations is 0-1:1.

[0011] In some embodiments of the above-described NK cell culture systems, the cytokines are selected from one or more interleukins.

[0012] In some embodiments, the interleukin is selected from one or more of IL-2, IL-3, IL-7, IL-12, IL-15, IL-18 and IL-21.

[0013] In some embodiments, the interleukin is IL-2, with a final concentration of 0-1000 IU / mL.

[0014] In one exemplary embodiment, the final concentration of IL-2 is 100 IU / mL.

[0015] In some embodiments, the basal culture medium is an immune cell expansion basal culture medium selected from one or more of the following: TBD-G (Tianjin Haoyang, #ANDL-TBD-G), X-VIVO15 (Lonza, #04-418Q), ALyS505NK-EX (BASO Beso Cells, #01400P10), ALyS505NK-AC (BASO Beso Cells, #01600P02), SCGM (CELL GENIX, #20802-0500), MEM α (GICBO, Thermo Fisher Scientific, #31985070), AIM V (GICBO, Thermo Fisher Scientific, #12055091), and self-prepared culture medium HDM-V2. The self-prepared culture medium HDM-V2 includes the following components: DMEM / F12, IMDM, lipid concentrate with clearly defined chemical composition, insulin-transferrin-seleno-aminoethanol, recombinant human serum albumin, vitamin C, and transferrin.

[0016] In some exemplary embodiments, the final concentrations of DMEM / F12, IMDM, lipid concentrate with clearly defined chemical composition, insulin-transferrin-seleno-aminoethanol, recombinant human serum albumin, vitamin C, and transferrin are 50%, 50%, 1%, 1%, 0-100 mg / mL, 0-100 μg / mL, and 0-100 μg / mL, respectively.

[0017] In one exemplary embodiment, the basal culture medium for immune cell expansion is the TBD-G medium.

[0018] On the other hand, the present invention also provides the application of the above-mentioned culture system in the culture of NK cells.

[0019] In another aspect, the present invention provides a method for expanding and culturing NK cells, which uses the above-described NK cell culture system to directly expand and culture NK cells from a cell population containing NK cells.

[0020] In one embodiment, the cell population containing NK cells is PBMCs.

[0021] In some exemplary embodiments, a method for expanding cultured NK cells includes the following steps:

[0022] a. The cell population containing NK cells is mixed with the feeder cells and inoculated into NK cell culture medium I for cell culture; b. The cell population containing NK cells obtained in a is mixed with the feeder cells and inoculated into NK cell culture medium II for cell culture; c. The cells obtained in b are harvested.

[0023] In some exemplary embodiments, the culture time for step a is 7 days; the culture time for step b is 7-28 days.

[0024] In one exemplary embodiment, the culture time for step a is 0-7 days; the culture time for step b is 0-28 days.

[0025] This invention also provides a method for directly expanding and culturing NK cells from PBMCs, comprising the following steps:

[0026] Day 0, 2×10 6 PBMCs of 5 × 10⁵ cells / mL and PBMCs of 5 × 10⁵ cells / mL 5 K562 cells were mixed and seeded into the NK cell culture medium I for cell culture.

[0027] Day 3, Counting, by 1×10 6 The cells, which were mixed and cultured in the previous step, were seeded into the NK cell culture medium I at a cell density of 1 cell / mL for cell culture.

[0028] Day 5, Counting, by 1×10 6 The cells cultured in the previous step were seeded into the NK cell culture medium I at a cell density of 1 cell / mL for cell culture.

[0029] Day 7, Counting, 1×10 6 PBMCs of 1 cell / mL and 2.5 × 105 K562 cells were mixed and seeded into the NK cell culture medium II for cell culture.

[0030] Count every other day, in increments of 1×10 6 The mixed cells were seeded into the NK cell culture medium II at a cell density of 1 cell / mL for cell culture.

[0031] NK cells were harvested between Day 14 and 35.

[0032] In a typical embodiment, the NK cell culture medium I comprises the basal culture medium, 100 IU / mL IL-2 and 1 μg / mL OK-432; the NK cell culture medium II comprises the basal culture medium and 100 IU / mL IL-2; the basal culture medium is the TBD-G culture medium. Beneficial effects

[0033] This invention discloses a method for expanding and culturing NK cells, which for the first time combines OK-432 with irradiated feeder cells, such as K562 cells, to expand and culture NK cells in vitro. Compared with expanding and culturing NK cells using OK-432 or irradiated feeder cells alone, the efficiency of expanding and culturing NK cells using the combination of OK-432 and irradiated feeder cells disclosed in this invention is significantly higher, and the purity of NK cells prepared from PBMCs of different donors is small and high.

[0034] In this article:

[0035] NK cells, or natural killer cells, originate from bone marrow lymphoid stem cells and are lymphotoxic cells. NK cells express CD56 on their cell surface but do not express CD3 (CD56). + CD3 -(Pfefferle A, et al., Frontiers In Immunology, 11:812, 2020). In this invention, there is no particular limitation on the NK cells used for in vitro cell culture. For example, NK cells may include one or more of the following: progenitor cells, embryonic stem cells, cells derived from embryonic stem cells, embryonic germ cells, cells derived from embryonic germ cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or immortalized cells; or NK cells isolated from peripheral blood, umbilical cord blood, lymph nodes, ascites, pleural effusion, thymus, tumors, or bone marrow. In some embodiments of this invention, NK cells or cell populations including NK cells are isolated from human peripheral blood or umbilical cord blood; in some embodiments of this invention, NK cells or cell populations including NK cells are derived from induced pluripotent stem cells.

[0036] "Basic Culture Medium": The basic culture medium used in this invention is not particularly limited in terms of its components, as long as it can provide cell nutrition and / or promote cell proliferation, activation and / or differentiation. For example, commercially available culture media TBD-G (Tianjin Haoyang, #ANDL-TBD-G), X-VIVO15 (LONZA, #04-418Q), ALYS505NK-EX (BASO Beso Cells, #01400P10), ALYS505NK-AC (BASO Beso Cells, #01600P02), SCGM (CELL GENIX, #20802-0500), MEM α (GICBO, Thermo Fisher Scientific, #31985070), AIM V (GICBO, Thermo Fisher Scientific, #12055091), etc., or self-prepared culture medium HDM-V2.In some embodiments of the present invention, a self-prepared culture medium, HDM-V2, is used. Its components include: DMEM / F12, IMDM, a lipid concentrate with clearly defined chemical composition, insulin-transferrin-selenoethanol, recombinant human serum albumin, vitamin C, and transferrin, with final concentrations of 50%, 50%, 1%, 1%, 0-100 mg / mL, 0-100 µg / mL, and 0-100 µg / mL, respectively. The final concentrations of each component of the self-prepared culture medium HDM-V2 are not particularly limited as long as they promote cell proliferation, activation, and / or differentiation. For example, the final concentration of recombinant human serum albumin can be 0 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, and 75 mg / mL. The final concentration of vitamin C can be 0 µg / mL, 80 µg / mL, 85 µg / mL, 90 µg / mL, 95 µg / mL, or 100 µg / mL, but is not limited to these values. Preferably, it is 0-100 µg / mL and not 0, more preferably 5 µg / mL. For example, the final concentration of vitamin C can be 0 µg / mL, 1 µg / mL, 5 µg / mL, 10 µg / mL, 15 µg / mL, 20 µg / mL, 25 µg / mL, 30 µg / mL, 35 µg / mL, 40 µg / mL, 45 µg / mL, 50 µg / mL, 55 µg / mL, 60 µg / mL, 65 µg / mL, 70 µg / mL, 75 µg / mL, 80 µg / mL, 85 µg / mL, 90 µg / mL, 95 µg / mL, or 100 µg / mL, but is not limited to these values. Preferably, it is 0-100 µg / mL and not 0, more preferably 70 µg / mL. µg / mL; for example, the final concentration of transferrin can be 0 µg / mL, 1 µg / mL, 5 µg / mL, 10 µg / mL, 15 µg / mL, 20 µg / mL, 25 µg / mL, 30 µg / mL, 35 µg / mL, 40 µg / mL, 45 µg / mL, 50 µg / mL, 55 µg / mL, 60 µg / mL, 65 µg / mL, 70 µg / mL, 75 µg / mL, 80 µg / mL, 85 µg / mL, 90 µg / mL, 95 µg / mL, 100 µg / mL, but is not limited thereto, preferably 0-100 µg / mL and not 0, more preferably 100 µg / mL.

[0037] "K562 cells": K562 cells are a cell line derived from chronic myeloid leukemia and are often used as feeder cells or induced cells to expand and activate natural killer (NK) cells. Their application plays an important role in the expansion and culture of NK cells, especially in the clinical and research development of NK cells as a means of tumor immunotherapy. In this invention, when using K562 cells as feeder cells to expand and culture NK cells in vitro, the ratio of K562 cells to NK cells or cell populations containing NK cells, such as PBMCs, is not particularly limited as long as it effectively promotes NK cell expansion and activation. For example, the ratio of PBMCs to K562 cells can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, but is not limited to these; preferably 1:1-10:1, more preferably 4:1.

[0038] OK-432: Its main component is low-toxicity Streptococcus pyogenes, which is inactivated after penicillin treatment. OK-432 is an immunomodulator initially used in cancer immunotherapy and the treatment of some infectious diseases. It can enhance the host's anti-tumor or anti-infective capabilities by stimulating the immune system. OK-432 can activate natural killer (NK) cells by interacting with immune cells such as dendritic cells, macrophages, and T cells, promoting the production of cytokines (such as IL-12, IL-18, and IFN-γ). In in vitro NK cell culture, OK-432 can be used as an immunostimulant to promote NK cell proliferation and enhanced activity. OK-432 induces dendritic cells to secrete cytokines by stimulating the Toll-like receptor (TLR) signaling pathway, which in turn promotes NK cell proliferation and functional enhancement. The concentration of 0K-432 used in this invention can be any concentration that can mediate the activation, proliferation, or differentiation of immune cells such as NK cells, for example, 0 µg / mL, 1 µg / mL, 2 µg / mL, 3 µg / mL, 4 µg / mL, 5 µg / mL, 6 µg / mL, 7 µg / mL, 8 µg / mL, 9 µg / mL, 10 µg / mL, but is not limited thereto; preferably 0-10 µg / mL and not 0; more preferably 1 µg / mL.

[0039] "Interleukin": Interleukin is a cytokine that mediates the activation, proliferation and differentiation of immune cells. The interleukins used in this invention are selected from one or more of IL-2, IL-3, IL-7, IL-12, IL-15, IL-18, and IL-21. Their concentrations are not particularly limited as long as they can mediate the activation, proliferation, and differentiation of immune cells such as NK cells. For example, the concentrations of IL-2 can be 1 IU / mL, 10 IU / mL, 20 IU / mL, 30 IU / mL, 40 IU / mL, 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 150 IU / mL, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, etc. The values ​​are IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, but not limited thereto; preferably 0-1000 IU / mL and not 0, more preferably 100 IU / mL.

[0040] All publications, documents, and patents mentioned herein are hereby incorporated in their entirety by reference, as are each publication, document, or patent not specifically and individually indicated to be incorporated herein by reference in its entirety. In case of conflict, this application (including any definitions herein) shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein shall not be construed as an admission or recommendation of any kind.

[0041] The section headings used in this article are for organizational purposes only and are not intended to limit the topics described. Attached Figure Description

[0042] Figure 1: Growth curve of PB-NK cells from the G01 donor expanded and cultured in Example 1;

[0043] Figure 2: Cell viability curve of PB-NK cells from the G01 donor in Example 1;

[0044] Figure 3: Flow cytometry results of the surface markers CD3, CD16, CD56 and CD335 of the PB-NK cells of the G01 donor detected on Day 16 in Example 1;

[0045] Figure 4: A bar chart comparing the relative expansion folds of PB-NK cells from G01 donors in Example 2, with and without OK-342 amplification culture.

[0046] Figure 5: The fold increase of PB-NK at different OK432 concentrations in Example 2;

[0047] Figure 6: A bar chart comparing the relative expansion fold of G01 donor PB-NK cells in Comparative Example 3, comparing K562-APC cells expanded and cultured with K562-APC cells expanded and cultured without irradiation.

[0048] Figure 7: Changes in PB-NK amplification with different K562 ratios in Example 3;

[0049] Figure 8: Growth curve of PB-NK cells from the G09 donor expanded and cultured in Example 4;

[0050] Figure 9: Cell viability curve of PB-NK cells from the G09 donor in Example 4;

[0051] Figure 10: Flow cytometry results of detecting surface markers CD3, CD16, CD56 and CD335 of PB-NK cells from the G09 donor on Day 15 in Example 4;

[0052] Figure 11: A bar chart comparing the killing efficiency of G09 donor PB-NK cells (expanded to Day 15) in killing cancer cells K562 cells, Jurkat cells and Nalm6 cells at different effector-target ratios E:T=5:1, 2:1 or 1:1 in Example 5.

[0053] Figure 12: A bar chart comparing the killing efficiency of PB-NK cells (expanded to Day 33) from G01 donors in killing cancer cells K562, Jurkat, and Nalm6 cells at different effector-target ratios E:T = 5:1, 2:1, or 1:1 in Example 6. Detailed Implementation

[0054] The following detailed description of the invention's concept and technical effects, in conjunction with specific embodiments, provides a clear and complete understanding of the invention's technical solution, the technical problems it solves, and its beneficial effects. Obviously, the described embodiments are only some, not all, of the embodiments of the invention; other embodiments obtained by those skilled in the art based on the embodiments of the invention without creative effort are all within the scope of protection of the invention.

[0055] Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions known in the art, or as selected according to the product instructions. Reagents and raw materials not specifically named in this invention are commercially available.

[0056] The K562-BL, Jurkat-BL, and Nalm6-BL cell lines used in the following examples are fluorescent reporter cell lines that overexpress BFP-luciferase. Example 1

[0057] 1. Methods for expanding and culturing NK cells from human PBMCs:

[0058] PBMCs were isolated from peripheral blood of healthy individuals (the isolation method is well known to those skilled in the art), and NK cells (PB-NK cells) were expanded and cultured from the PBMCs; the specific expansion method is as follows:

[0059] Day 0, 2×10 6 PBMCs of 5 × 10⁵ cells / mL and PBMCs of 5 × 10⁵ cells / mL 5 K562-APC cells were mixed and seeded into the NK cell culture medium I for cell culture; the NK cell culture medium I contained the TBD-G basal medium (Tianjin Haoyang, #ANDL-TBD-G), IL-2 at a final concentration of 100 IU / mL (Sihuan Bio / P763965) and 1 μg / mL OK-432 (Tongli Haiyuan / GMP-TL107-500 ug).

[0060] Day 3, Counting, by 1×10 6 The mixed cells were seeded into the NK cell culture medium I at a cell density of 1 cell / mL for cell culture.

[0061] Day 5, Counting, by 1×10 6 The mixed cells were seeded into the NK cell culture medium I at a cell density of 1 cell / mL for cell culture.

[0062] Day 7, Counting, 1×10 6 PBMCs of 1 cell / mL and 2.5 × 10 5 K562-APC cells / mL were mixed and seeded into the NK cell culture medium II for cell culture; the NK cell culture medium II contained the TBD-G basal medium and 100 IU / mL IL-2;

[0063] Count every other day, in increments of 1×10 6The mixed cells were seeded into NK cell culture medium II at a cell density of 10 cells / mL for cell culture, and NK cells were harvested on Day 14-35.

[0064] 2. Expanding PB-NK cells:

[0065] Day 0: Using the method described above for expanding and culturing PB-NK cells, 9.65 × 10⁶ cells were inoculated from healthy human donor 01 (G01). 6 A number of PBMCs were expanded, cultured, and counted. The expansion and growth curve of the PB-NK cells is shown in Figure 1.

[0066] The changes in cell viability during the expansion of PB-NK cells were detected, and the results are shown in Figure 2. As can be seen from Figure 2, the viability of the PB-NK cells remained above 85% after 2 weeks of expansion culture.

[0067] On Day 16, flow cytometry was used to detect the expression of markers such as CD16, CD56 and CD335 in the expanded cultured PB-NK cells. The results are shown in Figures 3A, B and C, respectively.

[0068] As shown in Figures 3A, B, and C, on Day 16, in the expanded cultured G01 PB-NK cells, CD3 - CD56 + The proportion of NK cells is approximately 95%; CD56 + CD16 + The proportion of NK cells was approximately 93%; CD56 + CD335 + The proportion of NK cells is approximately 95%; the method for expanding and culturing PB-NK cells disclosed in this invention can efficiently expand NK cells from PBMCs of G01. Example 2

[0069] 1. To investigate the effect of OK-432 use on the expansion of PB-NK cells:

[0070] Referring to the method for amplifying and culturing PB-NK cells in Example 1, NK cell culture medium I without OK-432 was used as the control, and the culture medium with OK-432 was used as the experimental group to amplify and culture G01 PBMCs.

[0071] After culturing to Day 15, the expansion efficiency of PBMCs and K562-APC mixed cells in the control group OK-432 was counted and statistically analyzed relative to the experimental group with added OK-432. The results are shown in Figure 4 (where the data of the control group OK-432 were set to 1).

[0072] As shown in Figure 4, the expansion efficiency of PBMCs and K562-APC mixed cells in the experimental group with OK-432 added to the culture medium was significantly better than that in the control group without OK-432 added to the culture medium.

[0073] 2. Effects of different dosages of OK-432 on PB-NK cell expansion:

[0074] Referring to the method for amplifying and culturing PB-NK cells in Example 1, the amount of OK-432 used in NK medium I of different groups was different, with final concentrations of 0 / 1 / 2 / 5 / 10 μg / mL.

[0075] Amplification up to Day 13 for CD56 counting, statistical analysis + CD3 - The NK cell expansion fold was measured, and the results are shown in Figure 5. Example 3

[0076] 1. To investigate the effect of using K562-APC feeder cells on PB-NK cell expansion:

[0077] Referring to the method for expanding and culturing PB-NK cells in Example 1, a control group was used that did not use K562-APC cells to mix with PBMCs throughout the process, while an experimental group was used that used K562-APC cells to expand and culture G01 PBMCs.

[0078] After culturing to Day 15, the expansion efficiency of PB-NK cells in the control group K562 was counted and statistically analyzed relative to the experimental group that used K562-APC cells to expand PB-NK cells. The results are shown in Figure 6 (where the data of the control group K562 were set to 1).

[0079] As shown in Figure 6, the experimental group using K562-APC cells to expand and culture PB-NK cells increased the cell expansion fold by 6-8 times, which was significantly better than the control group without K562-APC cells.

[0080] 2. Effects of different amounts of feeder cells on PB-NK cell expansion:

[0081] Following the method for expanding and culturing PB-NK cells from human PBMCs in Example 1, on Day 0, in different groups, the ratios of PBMC:K562-APC were 1:0 (i.e., no feeder cells added) and 1:0.25 (i.e., 2 × 10⁻⁵ cells added), respectively. 6 PBMCs per mL + 5 × 10 5 Irradiated K562-APC at a ratio of 1:0.5 (i.e., 2×10⁻⁶ cells / mL). 6 PBMCs per mL + 1 × 10 6irradiated K562-APC at a ratio of 1:1 (i.e., 2×10⁻⁶ cells / mL) 6 PBMCs / mL + 2×10 6 Irradiated K562-APC cells were added at a ratio of 1 cell / mL as feeder cells, and the remaining procedures were the same as in Example 1.

[0082] Amplification up to Day 13 for CD56 counting, statistical analysis + CD3 - The fold expansion of PB-NK cells was shown in Figure 7. Example 4

[0083] Take 1×10 from healthy human donor 09 (G09) 5 One PBMC was expanded and cultured according to the method for expanding and culturing NK cells in Example 1. The growth curve of PB-NK cells of G09 is shown in Figure 8.

[0084] As shown in Figure 8, on Day 15, the PB-NK cells of G09 expanded to approximately 1400-fold.

[0085] Figure 9 shows the cell viability of G09 PB-NK cells during the expansion process.

[0086] As shown in Figure 9, the cell viability of PB-NK cells was above 85% on Day 15.

[0087] Day 15; Flow cytometry was used to detect the expression of markers such as CD3, CD16, CD56 and CD335 in expanded cultured PB-NK cells. The results are shown in Figures 10A, B and C, respectively.

[0088] As shown in Figures 10A, B, and C, on Day 15, in PB-NK cells, CD3 - CD56 + The proportion of NK cells is approximately 95%; CD56 + CD16 + The proportion of NK cells was approximately 91%; CD56 + CD335 + The proportion of NK cells is approximately 86%; the method for expanding and culturing PB-NK cells disclosed in this invention can efficiently expand and culture NK cells from G09 PBMCs.

[0089] In conjunction with Example 1, the method for amplifying and culturing NK cells disclosed in this invention can also efficiently amplify and culture G01 PB-NK cells. It can be seen that by using the method for amplifying and culturing NK cells disclosed in this invention, the purity difference between PB-NK cells obtained from amplifying and culturing PB-NK cells from different donors is small, and the purity is high. Example 5

[0090] To detect the killing efficiency of G09 PB-NK cells in killing target cells.

[0091] In Example 4, PB-NK cells of G09 (expanded and cultured to Day 15) were used to kill cancer cells K562-BL cells, Jurkat (human T-cell lymphoblastic leukemia)-BL cells, and Nalm6 (human B-cell lymphoblastic leukemia)-BL cells at effector-to-target ratios of 5:1, 2:1, or 1:1, respectively. The specific method for detecting the killing efficiency was as follows:

[0092] G09 PB-NK cells were mixed with K562-BL cells, Jurkat-BL cells, and Nalm6-BL cells (9 groups in total) at effector-to-target ratios of 5:1, 2:1, or 1:1, respectively. Cells from each group were resuspended in basal medium (MEMα medium + 10% FBS) supplemented with 20 ng / mL IL-2 and 20 ng / mL IL-15. Three replicates of each group were seeded onto an ELISA plate. After 4 h of incubation, substrate was added, and chemiluminescence detection was performed using the ELISA plate to calculate the killing efficiency. The specific calculation method was as follows: wells containing only various cancer cells without PB-NK cells were used as reference wells, and their fluorescence value was the total fluorescence value of the original cancer cells. The remaining fluorescence value was based on the fluorescence value of the cancer cells remaining after PB-NK cell killing. The killing efficiency was calculated using the following formula: Killing efficiency (%) = (Total fluorescence value - Remaining fluorescence value) / Total fluorescence value × 100%;

[0093] Figure 11 shows the killing efficiency of G09 PB-NK cells in killing cancer cells in different groups under different effector-target ratios.

[0094] MEMα medium: Gibco, catalog number: #32571036; FBS: Gibco, catalog number: #10099148.

[0095] As shown in Figure 11, under different effector-to-target ratios E:T = 5:1, 2:1 or 1:1, G09 PB-NK cells (expanded to Day 15) can efficiently kill cancer cells K562 cells, Jurkat cells and Nalm6 cells. Example 6

[0096] To detect the killing efficiency of G01 PB-NK cells in killing target cells.

[0097] In Example 1, G01 PB-NK cells expanded and cultured to Day 33 were used. Following the method in Example 3 for detecting the killing efficiency of G09 PB-NK cells in killing cancer cells, G01 PB-NK cells were used to kill cancer cells K562-BL cells, Jurkat-BL cells, and Nalm6-BL cells respectively at effector-to-target ratios E:T=5:1, 2:1, or 1:1. The results are shown in Figure 12.

[0098] As shown in Figure 12, under different effector-target ratios E:T=5:1, 2:1 or 1:1, G01 PB-NK cells (expanded and cultured to Day 33) can still efficiently kill cancer cells K562 cells, Jurkat cells and Nalm6 cells.

Claims

1. An NK cell culture system, characterized by, The NK cell culture system includes feeder cells, NK cell culture medium I, and NK cell culture medium II; The trophoblast cells lose or have their proliferative capacity reduced due to irradiation. The NK cell culture medium I contains basal culture medium, OK-432, and cytokines; The NK cell culture medium II contains basal culture medium and cytokines.

2. The NK cell culture system of claim 1, wherein, The trophoblast cells include K562 cells, EBV-transformed lymphoblasts, PBMCs, and Wilms tumor cells.

3. The NK cell culture system of claim 2, wherein, The trophoblast cells are K562 cells; the K562 cells overexpress the 4-1BBL gene, the IL-15 gene, and the IL-21 gene.

4. The NK cell culture system of claim 1, wherein, The final concentration of OK-432 is 0-10 μg / mL.

5. The NK cell culture system of claim 1, wherein, The initial amount of the trophoblast cells added is: a. a concentration of 0-2 x 10 6 cells / mL; or, b. The ratio of the cultured cells to the cell population is 0-1:

1.

6. The NK cell culture system of claim 1, wherein The cytokines are selected from one or more interleukins.

7. The NK cell culture system of claim 6, wherein the NK cell culture system is a serum-free NK cell culture system. The interleukin is selected from one or more of IL-2, IL-3, IL-7, IL-12, IL-15, IL-18 and IL-21.

8. The NK cell culture system of claim 7, wherein the NK cell culture system is a serum-free NK cell culture system. The interleukin is IL-2, with a final concentration of 0-1000 IU / mL.

9. The NK cell culture system of claim 1, wherein, The basal culture medium is an immune cell expansion basal culture medium, which includes at least one of the following: TBD-G, X-VIVO15, ALyS505NK-EX, ALyS505NK-AC, SCGM, MEM α, AIM V and self-prepared culture medium HDM-V2. The self-prepared culture medium HDM-V2 includes the following components: DMEM / F12, IMDM, lipid concentrate with clearly defined chemical composition, insulin-transferrin-seleno-aminoethanol, recombinant human serum albumin, vitamin C, and transferrin.

10. The use of the NK cell culture system according to any one of claims 1-9 in culturing NK cells.

11. A method of expanding NK cells in culture, comprising, NK cells are directly expanded and cultured from a cell population containing NK cells using the NK cell culture system as described in any one of claims 1-9.

12. The method of claim 11 wherein, Includes the following steps: a. The cell population containing NK cells is mixed with the feeder cells and inoculated into the NK cell culture medium I for cell culture; b. Mix the cell population containing NK cells obtained in a with the feeder cells and inoculate them into the NK cell culture medium II for cell culture. c. Harvest the cells cultured in b; 13. The method of claim 12 wherein, The incubation period for step a is 0-7 days; the incubation period for step b is 0-28 days.

14. A method of expanding cultured NK cells directly from PBMCs, characterized in that, The steps are as follows: Day 0, 2x10 6 PBMCs and 5x10 5 cells / mL of K562 cells were mixed and inoculated into the NK cell culture medium I for cell culture; the NK cell culture medium I comprises the basic culture medium, 100 IU / mL IL-2 and 1 μg / mL OK432; Day 3, count, seed at 1 x 10 6 The cells mixed and cultured in the previous step are inoculated into the NK cell culture medium I at a cell density of 1 x 10 Day 5, count, seed at 1 x 10 6 The cells after culture in the previous step were inoculated into the NK cell culture medium I at a cell density of 1 x 10 Day 7, count, seed 1 x 10 6 PBMCs and 2.5 x 10 5 K562 cells / mL into the NK cell culture medium II for cell culture; the NK cell culture medium II comprises the basal medium and 100 IU / mL IL-2; The mixed cells are inoculated into the NK cell culture medium II at a cell density of 1 x 10 6 cells / mL every other day for cell culture. NK cells were harvested between Day 14 and 35.

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