A culture medium and culture method for promoting anti-tumor activity of NK cells

Abstract

The present invention relates to the technical field of in vitro cell culture. The present invention provides a culture medium and a culture method for promoting the anti-tumor activity of NK cells, the culture medium comprising a first culture medium and a second culture medium; the first culture medium is based on AIM-V culture medium and IL-2 and IL-21 are added; the second culture medium is based on AIM-V culture medium and IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid are added. The culture medium of the present invention can be used for in vitro culture of NK cells, can effectively improve the metabolism of NK cells, improve the self-activity, anti-tumor activity, ROS resistance of cultivated NK cells, and enhance immunosuppression resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro cell culture, and in particular to a culture medium and a culture method for promoting the anti-tumor activity of NK cells. Background Art

[0002] Natural killer cells (NK) 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 certain cases. They can recognize target cells and kill mediators.

[0003] NK cells are usually in a state of immune quiescence in the normal human body, accounting for about 5% to 15% of lymphocytes in the blood. The difficulties currently faced in the in vitro culture of NK cells include: 1. The higher the purity of the cells, the more difficult it is to expand; 2. Currently, genetically modified tumor cell lines K562 or EBV-transformed lymphoblastoid cell lines are mostly used to stimulate the rapid expansion of NK cells. However, the addition of these factors has brought concerns about the safety of cell products.

[0004] Therefore, it is very important to seek a NK cell culture method that can improve cell activity and have high anti-tumor activity. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium and a culture method for promoting the anti-tumor activity of NK cells, which are used for the in vitro culture of NK cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a culture medium for promoting the anti-tumor activity of NK cells, the culture medium comprising a first culture medium and a second culture medium;

[0008] The first culture medium is based on AIM-V culture medium and supplemented with IL-2 and IL-21;

[0009] The second culture medium is based on AIM-V culture medium and is supplemented with IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid.

[0010] Preferably, the concentration of IL-2 in the first culture medium is 1200-1500 IU / mL, and the concentration of IL-21 is 12-15 ng / mL.

[0011] Preferably, the concentration of IL-2 in the second culture medium is 700-800 IU / mL, the concentration of IL-21 is 6-8 ng / mL, the concentration of nicotinamide is 40-50 mM, the concentration of TGFβ1 inhibitor is 4-6 μM, and the concentration of the fatty acid is 0.1-0.3 mM.

[0012] Preferably, the fatty acids are arachidonic acid, myristic acid and palmitoleic acid, and the volume ratio of arachidonic acid, myristic acid and palmitoleic acid is 1:2.5-3.5:1.5-1.9.

[0013] The present invention also provides application of the culture medium in promoting the anti-tumor activity of NK cells.

[0014] The present invention also provides a method for cultivating NK cells using the culture medium, comprising the following steps:

[0015] (1) Inoculating NK cells into the first culture medium and culturing for 10 to 14 days;

[0016] (2) Replace the culture medium with the second culture medium and continue culturing for 16 to 25 days.

[0017] Preferably, the inoculation concentration of NK cells in step (1) is 6 to 8×10 5 Pieces / mL.

[0018] Preferably, the method of culturing in step (1) is as follows: adding the first culture medium once every 60 to 72 hours to keep the concentration of NK cells at 6 to 8×10 5 Pieces / mL.

[0019] Preferably, the method for continuing cultivation in step (2) is: replacing the culture medium every 36 to 48 hours, and when the concentration of NK cells reaches 1.8×10 6 When the concentration is above 100 / mL, add the second culture medium which accounts for 40-60% of the total volume.

[0020] The present invention provides a culture medium and a culture method for promoting the anti-tumor activity of NK cells, wherein the culture medium comprises a first culture medium and a second culture medium; the first culture medium is based on the AIM-V culture medium and IL-2 and IL-21 are added; the second culture medium is based on the AIM-V culture medium and IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid are added. The culture medium of the present invention can be used for in vitro culture of NK cells, can effectively improve the metabolism of NK cells, improve the self-activity, anti-tumor activity, ROS resistance of the cultivated NK cells, and enhance the resistance to immunosuppression. DETAILED DESCRIPTION

[0021] The present invention provides a culture medium for promoting the anti-tumor activity of NK cells, the culture medium comprising a first culture medium and a second culture medium;

[0022] The first culture medium is based on AIM-V culture medium and supplemented with IL-2 and IL-21;

[0023] The second culture medium is based on AIM-V culture medium and is supplemented with IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid.

[0024] In the present invention, the concentration of IL-2 in the first culture medium is preferably 1200 to 1500 IU / mL, more preferably 1350 IU / mL, and the concentration of IL-21 is preferably 12 to 15 ng / mL, more preferably 13 to 14 ng / mL.

[0025] In the present invention, the concentration of IL-2 in the second culture medium is preferably 700-800 IU / mL, more preferably 750 IU / mL, the concentration of IL-21 is preferably 6-8 ng / mL, more preferably 7 ng / mL, the concentration of nicotinamide is preferably 40-50 mM, more preferably 45 mM, the concentration of TGFβ1 inhibitor is preferably 4-6 μM, more preferably 5 μM, and the concentration of the fatty acid is preferably 0.1-0.3 mM, more preferably 0.2 mM.

[0026] In the present invention, the fatty acids are preferably arachidonic acid, myristic acid and palmitoleic acid, and the volume ratio of arachidonic acid, myristic acid and palmitoleic acid is preferably 1:2.5-3.5:1.5-1.9, and more preferably 1:3:1.7.

[0027] The present invention also provides application of the culture medium in promoting the anti-tumor activity of NK cells.

[0028] The present invention also provides a method for cultivating NK cells using the culture medium, comprising the following steps:

[0029] (1) Inoculating NK cells into the first culture medium and culturing for 10 to 14 days;

[0030] (2) Replace the culture medium with the second culture medium and continue culturing for 16 to 25 days.

[0031] In the present invention, the inoculation concentration of NK cells in step (1) is preferably 6 to 8×10 5 / mL, more preferably 7×10 5 Pieces / mL.

[0032] In the present invention, the cultivation method in step (1) is preferably: adding the first culture medium once every 60 to 72 hours to keep the concentration of NK cells at 6 to 8×10 5 The first culture medium is preferably added every 66 hours to maintain the concentration of NK cells at 6 to 8 × 10 5 Pieces / mL.

[0033] In the present invention, the method of continuing cultivation in step (2) is preferably: replacing the culture medium every 36 to 48 hours, and when the concentration of NK cells reaches 1.8×10 6 When the NK cell concentration reaches 1.8×10 / mL or more, add 40-60% of the total volume of the second culture medium, and more preferably replace the culture medium every 42 hours. 6 When the concentration of 50% of the total volume of the second culture medium was exceeded, 50% of the total volume of the second culture medium was added.

[0034] 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.

[0035] The AIM-V culture medium used in the examples of the present invention was purchased from Thermo Fisher Scientific.

[0036] Example 1

[0037] A method for cultivating NK cells, comprising the following steps:

[0038] (1) mixing arachidonic acid, myristic acid and palmitoleic acid in a volume ratio of 1:3.5:1.5 to obtain fatty acids;

[0039] (2) adding IL-2 and IL-21 to the AIM-V medium so that the concentration of IL-2 is 1200 IU / mL and the concentration of IL-21 is 15 ng / mL to obtain a first medium;

[0040] IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid are added to the AIM-V medium, so that the concentration of IL-2 is 700 IU / mL, the concentration of IL-21 is 8 ng / mL, the concentration of nicotinamide is 40 mM, the concentration of TGFβ1 inhibitor is 6 μM, and the concentration of the fatty acid is 0.3 mM, to obtain a second medium;

[0041] (3) NK cells were divided into 8×10 5 The first culture medium was inoculated at a ratio of 10 / mL and cultured for 14 days. During the culture process, the first culture medium was added once every 72 hours to reduce the concentration of NK cells to 8×10 5 Pieces / mL;

[0042] (4) The culture medium was then replaced with the second culture medium and cultured for another 16 days. The culture medium was replaced every 36 hours during the culture process. When the concentration of NK cells reached 1.8×10 6 When the concentration of 50 μg / mL or more was reached, the second culture medium was added at 40% of the total volume.

[0043] Example 2

[0044] A method for cultivating NK cells, comprising the following steps:

[0045] (1) mixing arachidonic acid, myristic acid and palmitoleic acid in a volume ratio of 1:2.5:1.9 to obtain fatty acids;

[0046] (2) adding IL-2 and IL-21 to the AIM-V medium so that the concentration of IL-2 is 1500 IU / mL and the concentration of IL-21 is 12 ng / mL to obtain a first medium;

[0047] IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid are added to the AIM-V medium, so that the concentration of IL-2 is 800 IU / mL, the concentration of IL-21 is 6 ng / mL, the concentration of nicotinamide is 50 mM, the concentration of TGFβ1 inhibitor is 4 μM, and the concentration of the fatty acid is 0.1 mM, to obtain a second medium;

[0048] (3) NK cells were divided into 6×10 5 The first culture medium was inoculated at a ratio of 10 / mL and cultured for 10 days. The first culture medium was added every 60 hours during the culture process to reduce the concentration of NK cells to 6×10 5 Pieces / mL;

[0049] (4) The culture medium was then replaced with the second culture medium and cultured for another 25 days. The culture medium was replaced every 48 hours during the culture process. When the concentration of NK cells reached 1.8×10 6 When the concentration of 50 μg / mL or more was reached, the second culture medium was added at 60% of the total volume.

[0050] Example 3

[0051] A method for cultivating NK cells, comprising the following steps:

[0052] (1) mixing arachidonic acid, myristic acid and palmitoleic acid in a volume ratio of 1:3:1.7 to obtain fatty acids;

[0053] (2) adding IL-2 and IL-21 to the AIM-V medium so that the concentration of IL-2 is 1350 IU / mL and the concentration of IL-21 is 14 ng / mL to obtain a first medium;

[0054] Adding IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid to the AIM-V medium, so that the concentration of IL-2 is 750 IU / mL, the concentration of IL-21 is 7 ng / mL, the concentration of nicotinamide is 45 mM, the concentration of TGFβ1 inhibitor is 5 μM, and the concentration of the fatty acid is 0.2 mM, to obtain a second medium;

[0055] (3) NK cells were divided into 7×10 5 The first culture medium was inoculated at a ratio of 10 / mL and cultured for 12 days. The first culture medium was added once every 66 hours during the culture process to reduce the concentration of NK cells to 7×10 5 Pieces / mL;

[0056] (4) The culture medium was then replaced with the second culture medium and cultured for another 20 days. The culture medium was replaced every 42 hours during the culture process. When the concentration of NK cells reached 1.8×10 6 When the concentration of 50% of the total volume of the second culture medium was exceeded, 50% of the total volume of the second culture medium was added.

[0057] Comparative Example

[0058] A method for cultivating NK cells, comprising the following steps:

[0059] (1) adding IL-2 and IL-21 to the AIM-V medium so that the concentration of IL-2 is 1350 IU / mL and the concentration of IL-21 is 14 ng / mL to obtain a first medium;

[0060] Adding IL-2 and IL-21 to the AIM-V medium to adjust the concentration of IL-2 to 750 IU / mL and the concentration of IL-21 to 7 ng / mL, to obtain a second medium;

[0061] (3) NK cells were divided into 7×10 5 The first culture medium was inoculated at a ratio of 10 / mL and cultured for 12 days. The first culture medium was added once every 66 hours during the culture process to reduce the concentration of NK cells to 7×10 5 Pieces / mL;

[0062] (4) The culture medium was then replaced with the second culture medium and cultured for another 20 days. The culture medium was replaced every 42 hours during the culture process. When the concentration of NK cells reached 1.8×10 6 When the concentration of 50% of the total volume of the second culture medium was exceeded, 50% of the total volume of the second culture medium was added.

[0063] Test example

[0064] (1) NK cells were cultured using the methods of Examples 1 to 3 and the comparative example, and the cell purity and expansion multiple were detected after the culture was completed.

[0065] (2) The NK cells cultured in Examples 1 to 3 and the comparative example were mixed with tumor cells K562 at a ratio of 3:1, and the killing effect of the NK cells cultured in each group on K562 tumor cells was determined by the lactate dehydrogenase release method.

[0066] result

[0067] (1) Flow cytometry was used to detect cell purity and expansion multiples, and the results showed:

[0068] The cell purity of NK cells obtained by the culture method of Example 1 was 94.7%, and the expansion multiple was 891.3 times;

[0069] The cell purity of NK cells obtained by the culture method of Example 2 was 93.1%, and the expansion multiple was 798.3 times;

[0070] The cell purity of NK cells obtained by the culture method of Example 3 was 96.2%, and the expansion multiple was 910.3 times;

[0071] The cell purity of NK cells obtained by the culture method of the comparative example was 87.8%, and the expansion multiple was 434.7 times.

[0072] (2) The lactate dehydrogenase release method was used to determine the killing effect of NK cells on K562 tumor cells. The results showed that:

[0073] The cell killing rate of the NK cells obtained by the culture method of Example 1 was 52.76% when the effector-target ratio was 5:1, 69.73% when the effector-target ratio was 10:1, 89.11% when the effector-target ratio was 20:1, and 100% when the effector-target ratio was 40:1;

[0074] The cell killing rate of NK cells obtained by the culture method of Example 2 was 53.81% when the effector-target ratio was 5:1, 67.09% when the effector-target ratio was 10:1, 86.10% when the effector-target ratio was 20:1, and 100% when the effector-target ratio was 40:1;

[0075] The cell killing rate of the NK cells obtained by the culture method of Example 3 was 54.09% when the effector-target ratio was 5:1, 70.21% when the effector-target ratio was 10:1, 90.35% when the effector-target ratio was 20:1, and 100% when the effector-target ratio was 40:1;

[0076] The cell killing rate of NK cells obtained by the comparative culture method was 31.04% when the effector-target ratio was 5:1, 53.87% when the effector-target ratio was 10:1, 78.06% when the effector-target ratio was 20:1, and 88.55% when the effector-target ratio was 40:1.

[0077] As can be seen from the above embodiments, the present invention provides a culture medium and a culture method for promoting the anti-tumor activity of NK cells, the culture medium comprising a first culture medium and a second culture medium; the first culture medium is based on the AIM-V culture medium and IL-2 and IL-21 are added; the second culture medium is based on the AIM-V culture medium and IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid are added. The culture medium of the present invention can be used for the in vitro culture of NK cells, can effectively improve the metabolism of NK cells, improve the self-activity, anti-tumor activity, and ROS resistance of the cultured NK cells, and enhance the resistance to immunosuppression.

[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 culture medium for promoting the anti-tumor activity of NK cells, characterized in that: The culture medium comprises a first culture medium and a second culture medium; The first culture medium is based on AIM-V culture medium and supplemented with IL-2 and IL-21; The second culture medium is based on AIM-V culture medium and supplemented with IL-2, IL-21, nicotinamide, TGFβ1 inhibitor and fatty acid; The concentration of IL-2 in the first culture medium is 1200-1500 IU / mL, and the concentration of IL-21 is 12-15 ng / mL; The concentration of IL-2 in the second culture medium is 700-800 IU / mL, the concentration of IL-21 is 6-8 ng / mL, the concentration of nicotinamide is 40-50 mM, the concentration of TGFβ1 inhibitor is 4-6 μM, and the concentration of the fatty acid is 0.1-0.3 mM; The fatty acids are arachidonic acid, myristic acid and palmitoleic acid, and the volume ratio of arachidonic acid, myristic acid and palmitoleic acid is 1:2.5-3.5:1.5-1.

9.

2. Use of the culture medium according to claim 1 in promoting the anti-tumor activity of NK cells.

3. A method for cultivating NK cells using the culture medium according to claim 1, characterized in that: The steps include: (1) Inoculating NK cells into the first culture medium and culturing for 10 to 14 days; (2) Replace the culture medium with the second culture medium and continue culturing for 16 to 25 days.

4. The method according to claim 3, characterized in that: The inoculation concentration of NK cells in step (1) is 6 to 8 × 10 5 Pieces / mL.

5. The method according to claim 4, characterized in that The method of cultivation in step (1) is: adding the first culture medium once every 60 to 72 hours to keep the concentration of NK cells at 6 to 8×10 5 Pieces / mL.

6. The method according to any one of claims 3 to 5, characterized in that: The method for continuing the cultivation in step (2) is: replace the culture medium every 36 to 48 hours, and when the concentration of NK cells reaches 1.8×10 6 When the concentration is above 100 / mL, add the second culture medium which accounts for 40-60% of the total volume.