Enhanced NK cell and preparation method thereof

Abstract

The present invention relates to the technical field of immune cell culture. The present invention provides an enhanced NK cell and a preparation method thereof, comprising the following steps: (1) inoculating mononuclear cells into a first culture medium, continuously culturing for 15 to 24 days, and isolating and obtaining NK cells; (2) inoculating NK cells into a second culture medium, and cultivating for 10 to 12 days; (3) replacing the culture medium with a third culture medium, and continuing to cultivate for 20 to 25 days to obtain the enhanced NK cell. The present invention improves the existing NK cell cultivation method and further cultivates the cultivated NK cells, thereby effectively improving the activity of NK cells and improving the killing performance of tumor cells.

Description

Technical Field

[0001] The present invention relates to the technical field of immune cell culture, and in particular to an enhanced NK cell and a preparation method thereof. Background Art

[0002] Human NK cells are isolated from human peripheral blood mononuclear cells; NK cells, or natural killer cells, are important immune cells in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity reactions and autoimmune diseases in some cases, and can recognize target cells and killing mediators. NK cells are derived from bone marrow lymphoid stem cells. Their differentiation and development depend on the bone marrow and thymus microenvironment. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes. NK cells are different from T and B cells. They are a type of lymphocytes that can non-specifically kill tumor cells and virus-infected cells without prior sensitization. Since the killing activity of NK cells is not restricted by MHC and does not depend on antibodies, it is called natural killer activity. NK cells have rich cytoplasm and contain large azure granules. The content of granules is positively correlated with the killing activity of NK cells. After NK cells act on target cells, the killing effect appears early, and the killing effect can be seen in 1 hour in vitro and 4 hours in vivo. The target cells of NK cells mainly include certain tumor cells (including some cell lines), virus-infected cells, certain autologous tissue cells (such as blood cells), parasites, etc. Therefore, NK cells are important immune factors for the body's anti-tumor and anti-infection, and are also involved in type II hypersensitivity reactions and graft-versus-host reactions.

[0003] Whether NK cells can play a role in tumor treatment depends on the number of cells and their ability to kill tumor cells. However, the proliferation rate of NK cells in vitro is limited, and the number is difficult to meet the needs. Therefore, how to improve the killing ability of cells in a limited number is an effective way to improve the effect of NK cells. Summary of the invention

[0004] The purpose of the present invention is to provide an enhanced NK cell and a preparation method thereof, which effectively improves the killing activity of NK cells by adding specific components and adjusting the culture method during the NK cell culture process.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing enhanced NK cells, comprising the following steps:

[0007] (1) inoculating mononuclear cells into the first culture medium, culturing continuously for 15 to 24 days, and isolating NK cells;

[0008] (2) inoculating NK cells into the second culture medium and culturing for 10 to 12 days;

[0009] (3) The culture medium is replaced with the third culture medium, and the culture is continued for 20 to 25 days to obtain the enhanced NK cells.

[0010] Preferably, the density of mononuclear cells inoculated in step (1) is 2 to 4×10 6 Pieces / mL.

[0011] Preferably, in step (1), the first culture medium is based on RPMI-1640 culture medium and contains the following raw materials at the following concentrations: human albumin 20-30 μg / mL, cyclohexane hexol 30-40 μg / mL, IL-26 0-80 μg / mL, and CD16 antibody 2-4 μg / mL.

[0012] Preferably, the culture medium is replaced every 36 to 60 hours during the continuous culture in step (1), and the cell density is adjusted to 2 to 4×10 6 Pieces / mL.

[0013] Preferably, the density of NK cells inoculated in step (2) is 0.6 to 1×10 6 Pieces / mL.

[0014] Preferably, the second culture medium in step (2) is based on RPMI-1640 culture medium and contains the following raw materials at the following concentrations: IL-26 0-80 μg / mL, IL-216-8 ng / mL, and Codonopsis pilosula I 30-40 μM.

[0015] Preferably, the culture medium is replaced every 36 to 60 hours during the cultivation in step (2), and the cell density is adjusted to 0.6 to 1×10 6 Pieces / mL.

[0016] Preferably, the third culture medium in step (3) is based on AIM-V culture medium and contains the following raw materials at the following concentrations: IL-26 0-80 μg / mL, IL-216-8 ng / mL, nicotinamide 40-50 mM, and tws119 30-40 μM.

[0017] Preferably, the culture medium is replaced every 36 to 60 hours during the cultivation in step (3), and the cell density is adjusted to 0.6 to 1×10 6 Pieces / mL.

[0018] The present invention also provides enhanced NK cells prepared by the preparation method.

[0019] The present invention provides an enhanced NK cell and a preparation method thereof, comprising the following steps: (1) inoculating mononuclear cells into a first culture medium, continuously culturing for 15 to 24 days, and isolating NK cells; (2) inoculating NK cells into a second culture medium, and culturing for 10 to 12 days; (3) replacing the culture medium with a third culture medium, and continuing to cultivate for 20 to 25 days to obtain the enhanced NK cell. The present invention improves the existing NK cell cultivation method and further cultivates the cultivated NK cells, thereby effectively improving the activity of NK cells and improving the killing performance of tumor cells. DETAILED DESCRIPTION

[0020] The present invention provides a method for preparing enhanced NK cells, comprising the following steps:

[0021] (1) inoculating mononuclear cells into the first culture medium, culturing continuously for 15 to 24 days, and isolating NK cells;

[0022] (2) inoculating NK cells into the second culture medium and culturing for 10 to 12 days;

[0023] (3) The culture medium is replaced with the third culture medium, and the culture is continued for 20 to 25 days to obtain the enhanced NK cells.

[0024] In the present invention, the density of mononuclear cells inoculated in step (1) is preferably 2 to 4×10 6 / mL, more preferably 3×10 6 Pieces / mL.

[0025] In the present invention, the first culture medium in step (1) is preferably based on RPMI-1640 culture medium, containing the following raw materials at the following concentrations: human albumin 20-30 μg / mL, cyclohexane hexol 30-40 μg / mL, IL-260-80 μg / mL, CD16 antibody 2-4 μg / mL, and more preferably: human albumin 25 μg / mL, cyclohexane hexol 35 μg / mL, IL-270 μg / mL, CD16 antibody 3 μg / mL.

[0026] In the present invention, the culture medium is preferably replaced every 36 to 60 hours during the continuous culture in step (1), and more preferably every 48 hours. When replacing the culture medium, the cell density is preferably adjusted to 2 to 4×10 6 / mL, more preferably 3×10 6 Pieces / mL.

[0027] In the present invention, the density of NK cell inoculation in step (2) is preferably 0.6 to 1×10 6 pcs / mL, more preferably 0.8×10 6Pieces / mL.

[0028] In the present invention, the second culture medium in step (2) is preferably based on RPMI-1640 culture medium, containing the following concentrations of raw materials: IL-260~80μg / mL, IL-216~8ng / mL, Codonopsis pilosula I 30~40μM, and more preferably: IL-270μg / mL, IL-217ng / mL, Codonopsis pilosula I 35μM.

[0029] In the present invention, the culture medium is preferably replaced every 36 to 60 hours during the cultivation in step (2), and more preferably every 48 hours. When replacing the culture medium, the cell density is preferably adjusted to 0.6 to 1×10 6 pcs / mL, more preferably 0.8×10 6 Pieces / mL.

[0030] In the present invention, the third culture medium in step (3) is preferably based on AIM-V culture medium, and contains the following raw materials at the following concentrations: IL-260-80 μg / mL, IL-216-8 ng / mL, nicotinamide 40-50 mM, tws11930-40 μM, and more preferably: IL-270 μg / mL, IL-217 ng / mL, nicotinamide 45 mM, tws11935 μM.

[0031] In the present invention, the culture medium is preferably replaced every 36 to 60 hours during the cultivation in step (3), and more preferably every 48 hours. When replacing the culture medium, the cell density is preferably adjusted to 0.6 to 1×10 6 pcs / mL, more preferably 0.8×10 6 Pieces / mL.

[0032] The present invention also provides enhanced NK cells prepared by the preparation method.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] A method for preparing enhanced NK cells comprises the following steps:

[0036] 1. Culture medium preparation:

[0037] (1) Based on RPMI-1640 medium, a first medium was prepared according to the standard of 30 μg / mL human albumin, 40 μg / mL cyclohexane hexol, 60 μg / mL IL-2, and 4 μg / mL CD16 antibody, and set aside;

[0038] (2) Based on RPMI-1640 medium, a second medium was prepared according to the standard of IL-2 60 μg / mL, IL-2 18 ng / mL, and Codonopsis pilosula Ⅰ 40 μM for use;

[0039] (3) Based on AIM-V medium, a third medium was prepared according to the standard of IL-2 60 μg / mL, IL-2 16 ng / mL, nicotinamide 50 mM, and TWS119 40 μM, and set aside.

[0040] 2. NK cell culture

[0041] (1) Mononuclear cells were divided into 2×10 6 The cells were inoculated into the first culture medium at a density of 4 × 10 / mL and cultured continuously for 24 days. The culture medium was replaced every 60 hours during the culture period. The cell density was adjusted to 4 × 10 6 / mL, and NK cells were obtained after the culture was completed;

[0042] (2) NK cells were divided into 1×10 6 The cells were inoculated into the second culture medium at a density of 1×10 / mL and cultured for 10 days. The culture medium was replaced every 60 hours during the culture period. The cell density was adjusted to 1×10 6 Pieces / mL;

[0043] (3) The culture medium was replaced with the third culture medium and the culture was continued for 20 days. The culture medium was replaced every 60 hours during the culture period. When the culture medium was replaced, the cell density was adjusted to 1×10 6 / mL, and the enhanced NK cells are obtained after the culture is completed.

[0044] Example 2

[0045] A method for preparing enhanced NK cells comprises the following steps:

[0046] 1. Culture medium preparation:

[0047] (1) Based on RPMI-1640 medium, a first medium was prepared according to the standard of 20 μg / mL human albumin, 30 μg / mL cyclohexane hexol, 80 μg / mL IL-2, and 2 μg / mL CD16 antibody, and set aside;

[0048] (2) Based on RPMI-1640 medium, a second medium was prepared according to the standard of IL-2 80 μg / mL, IL-2 16 ng / mL, and Codonopsis pilosula I 30 μM for use;

[0049] (3) Based on AIM-V medium, a third medium was prepared according to the standard of IL-2 80 μg / mL, IL-2 18 ng / mL, nicotinamide 40 mM, and TWS119 30 μM, and set aside.

[0050] 2. NK cell culture

[0051] (1) Mononuclear cells were divided into 4 × 10 6 The cells were inoculated into the first culture medium at a density of 4 × 10 / mL and cultured continuously for 15 days. The culture medium was replaced every 36 hours during the culture period. The cell density was adjusted to 4 × 10 6 / mL, and NK cells were obtained after the culture was completed;

[0052] (2) NK cells were cultured at a rate of 0.6×10 6 The cells were inoculated into the second culture medium at a density of 0.6 × 10 / mL and cultured for 12 days. The culture medium was replaced every 36 hours during the culture period. The cell density was adjusted to 0.6 × 10 6 Pieces / mL;

[0053] (3) The culture medium was replaced with the third culture medium and the culture was continued for 25 days. The culture medium was replaced every 36 hours during the culture period. When the culture medium was replaced, the cell density was adjusted to 0.6×10 6 / mL, and the enhanced NK cells are obtained after the culture is completed.

[0054] Example 3

[0055] A method for preparing enhanced NK cells comprises the following steps:

[0056] 1. Culture medium preparation:

[0057] (1) Based on RPMI-1640 medium, a first medium was prepared according to the standard of 25 μg / mL human albumin, 35 μg / mL cyclohexane hexol, 70 μg / mL IL-2, and 3 μg / mL CD16 antibody, and set aside;

[0058] (2) Based on RPMI-1640 medium, a second medium was prepared according to the standard of IL-2 70 μg / mL, IL-2 17 ng / mL, and Codonopsis pilosula Ⅰ 35 μM for use;

[0059] (3) Based on AIM-V medium, a third medium was prepared according to the standard of IL-2 70 μg / mL, IL-2 17 ng / mL, nicotinamide 45 mM, and TWS119 35 μM, and set aside.

[0060] 2. NK cell culture

[0061] (1) Mononuclear cells were divided into 3×106 The cells were inoculated into the first culture medium at a density of 3 × 10 / mL and cultured continuously for 20 days. The culture medium was replaced every 48 hours during the culture period. The cell density was adjusted to 3 × 10 6 / mL, and NK cells were obtained after the culture was completed;

[0062] (2) NK cells were cultured at a rate of 0.8×10 6 The cells were inoculated into the second culture medium at a density of 0.8 × 10 / mL and cultured for 11 days. The culture medium was replaced every 48 hours during the culture period. The cell density was adjusted to 0.8 × 10 6 Pieces / mL;

[0063] (3) The culture medium was replaced with the third culture medium and the culture was continued for 22 days. The culture medium was replaced every 48 hours during the culture period. When the culture medium was replaced, the cell density was adjusted to 0.8×10 6 / mL, and the enhanced NK cells are obtained after the culture is completed.

[0064] Comparative Example 1

[0065] Take the NK cells cultured in step (1) of step 2 of Example 3 as the test object and set aside.

[0066] Comparative Example 2

[0067] Commercially available NK cells were selected for enhanced culture, and the remaining steps were the same as steps (2) and (3) in step 2 of Example 3. The culture medium used was the same as the second culture medium and the third culture medium of Example 3.

[0068] Comparative Example 3

[0069] The codonopsis pilosula I in the second culture medium and the nicotinamide and tws119 in the third culture medium were removed, and the remaining conditions were the same as those in Example 3.

[0070] Test example

[0071] The enhanced NK cells cultured in Examples 1 to 3 were taken and the cell density was adjusted to 1×10 6 / mL, numbered as test group samples 1 to 3; the cells obtained in comparative examples 1 to 3 were taken and the cell density was adjusted to 1×10 6 / mL, numbered as control group samples 1 to 3.

[0072] The test group samples 1-3 and the control group samples 1-3 were co-cultured with K562 tumor cells for 4 hours, and the killing effect was detected by lactate dehydrogenase release method. The results are shown in Table 1.

[0073] Table 1 Killing effect test results

[0074]

[0075]

[0076] The results showed that the enhanced NK cells cultivated by the cultivation method of the present invention effectively increased the activity of NK cells and improved the killing performance against tumor cells.

[0077] It can be seen from the above embodiments that the present invention provides an enhanced NK cell and a preparation method thereof, comprising the following steps: (1) inoculating mononuclear cells into a first culture medium, culturing continuously for 15 to 24 days, and isolating NK cells; (2) inoculating NK cells into a second culture medium, and cultivating for 10 to 12 days; (3) replacing the culture medium with a third culture medium, and continuing to cultivate for 20 to 25 days to obtain the enhanced NK cell. The present invention improves the existing NK cell cultivation method and further cultivates the cultivated NK cells, thereby effectively improving the activity of NK cells and improving the killing performance of tumor cells.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing enhanced NK cells, characterized in that: The steps include: (1) inoculating mononuclear cells into the first culture medium, culturing continuously for 15 to 24 days, and isolating NK cells; (2) inoculating NK cells into the second culture medium and culturing for 10 to 12 days; (3) replacing the culture medium with a third culture medium and continuing to culture for 20 to 25 days to obtain the enhanced NK cells; In step (1), the first culture medium is based on RPMI-1640 culture medium and contains the following raw materials at the following concentrations: human albumin 20-30 μg / mL, cyclohexane hexol 30-40 μg / mL, IL-2 60-80 μg / mL, and CD16 antibody 2-4 μg / mL; In step (2), the second culture medium is based on RPMI-1640 culture medium and contains the following raw materials at the following concentrations: IL-2 60-80 μg / mL, IL-21 6-8 ng / mL, and Codonopsis pilosula I 30-40 μM; The third culture medium in step (3) is based on AIM-V culture medium and contains the following raw materials at the following concentrations: IL-2 60-80 μg / mL, IL-21 6-8 ng / mL, nicotinamide 40-50 mM, and tws119 30-40 μM.

2. The preparation method according to claim 1, characterized in that: The density of mononuclear cells inoculated in step (1) is 2 to 4×10 6 Pieces / mL.

3. The preparation method according to claim 1, characterized in that: During the continuous culture in step (1), the culture medium is replaced every 36 to 60 hours, and the cell density is adjusted to 2 to 4 × 10 6 Pieces / mL.

4. The preparation method according to claim 1, characterized in that: The density of NK cell inoculation in step (2) is 0.6-1×10 6 Pieces / mL.

5. The preparation method according to claim 1, characterized in that: During the cultivation in step (2), the culture medium was replaced every 36 to 60 hours, and the cell density was adjusted to 0.6 to 1×10 6 Pieces / mL.

6. The preparation method according to claim 1, characterized in that: During the cultivation in step (3), the culture medium was replaced every 36 to 60 hours, and the cell density was adjusted to 0.6 to 1×10 6 Pieces / mL.

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