A method for improving NK cell killing activity

Abstract

The present invention relates to the technical field of NK cell culture, and in particular to a method for improving the killing activity of NK cells. The present invention provides a method for improving the killing activity of NK cells, comprising the following steps: (1) separating peripheral blood to obtain PBMC cells; (2) separating the PBMC cells to obtain NK cells; (3) inoculating the NK cells in an NK cell culture medium for culture. The method of the present invention can rapidly and massively amplify NK cells while ensuring a high viability of NK cells, thereby obtaining NK cells with high purity and high killing activity.

Description

Technical Field

[0001] The present invention relates to the technical field of NK cell culture, and in particular to a method for improving the killing activity of NK cells. Background Art

[0002] Natural killer cells (NK cells), a type of lymphocyte alongside T and B cells, are a key component of the innate immune system. In addition to participating in the innate immune response, NK cells also play a crucial regulatory role in adaptive immune regulation. Related research has found that NK cells can play a major role in a variety of pathological processes, including pathogenic microbial infections, autoimmune diseases, and tumors.

[0003] NK cells directly recognize target cells primarily through binding to MHC class I molecules on their surface receptors, without the involvement of antigen-presenting cells. When activated, NK cells can kill target cells through cytotoxicity and secrete cytokines such as interleukin-2 and interferon-γ to activate other effector lymphocytes.

[0004] NK cells only account for approximately 10% of peripheral blood lymphocytes, far from meeting clinical needs. Currently, NK cells are primarily expanded using cytokine stimulation, but the NK cells expanded by these methods still fall short of clinical needs. Furthermore, for further use of NK cells, their cytotoxic activity still needs to be improved. Therefore, finding methods to enhance NK cell cytotoxicity will help better utilize NK cells and potentially apply them to the treatment of autoimmune diseases, tumors, and other diseases. Summary of the Invention

[0005] The object of the present invention is to provide a method for improving the killing activity 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 method for improving the killing activity of NK cells, comprising the following steps:

[0008] (1) Separate peripheral blood to obtain PBMC cells;

[0009] (2) separating the PBMC cells to obtain NK cells;

[0010] (3) inoculating NK cells into NK cell culture medium for culture;

[0011] The NK cell culture medium comprises: 1-2 g / L L-lysine, 0.2-0.6 g / L methionine, 1-2 g / L L-tryptophan, 1-2 g / L L-proline, 6-10 g / L glutathione, 5-9 g / L coenzyme 1, 1-2 g / L silk peptide powder, 60-70 mL / L human plasma, 8-12 g / L human serum albumin, 0.16-0.18 μL / mL interleukin-10, 0.2-0.4 μL / mL interleukin-13, and 0.3-0.5 μL / mL interleukin-18, and the rest is basal culture medium.

[0012] Preferably, the basal culture medium is Lonza X-VIVO 15 serum-free culture medium, SCGM serum-free culture medium or Takara NK cell serum-free culture medium.

[0013] Preferably, the culture temperature is 37-38° C., and the volume concentration of CO 2 is 5%-6%.

[0014] Preferably, the culture time is 13 to 15 days.

[0015] Preferably, the inoculated cell density is 7 to 9×10 5 pieces / mL.

[0016] Preferably, during the culture process, the early rehydration medium is added every 2 to 3 days from the 0th to the 7th day of culture, and the late rehydration medium is added every 2 to 3 days thereafter; after rehydration, the cell density is always maintained at 7 to 9×10 5 pieces / mL.

[0017] Preferably, the early rehydration culture medium comprises: 60-70 mL / L human plasma, 0.16-0.18 μL / mL interleukin-10, 0.2-0.4 μL / mL interleukin-13 and 0.3-0.5 μL / mL interleukin-18, and the rest is basal culture medium.

[0018] Preferably, the late rehydration culture medium comprises: 0.2-0.3 μL / mL interleukin-10, 0.5-0.6 μL / mL interleukin-13 and 0.6-0.7 μL / mL interleukin-18, and the rest is basal culture medium.

[0019] The present invention also provides the use of NK cells prepared by the method for improving the killing activity of NK cells in the preparation of anti-tumor drugs.

[0020] Preferably, the tumor is non-small cell lung cancer, chronic myeloid leukemia, colorectal cancer or breast cancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Cultivating NK cells in a culture medium containing interleukins can produce NK cells with significantly improved killing activity. The present invention uses three interleukins: interleukin-10, interleukin-13, and interleukin-18 in combination, which significantly improves the killing activity of NK cells. However, if other types of interleukins are used instead, the effect is far inferior to that of the present invention.

[0023] The amino acids used in the present invention are all non-polar amino acids, which can effectively shorten the hysteresis phase of NK cell growth, thereby enabling rapid expansion and passage. The silk peptide powder added to the culture medium has a protective effect, which can alleviate the effects of external stimuli on mycoplasma growth. Glutathione can protect cells from oxidative stress and help form a favorable redox environment inside and outside the cells, which also helps shorten the hysteresis phase of NK cell growth.

[0024] The culture medium provided by the present invention also contains NK cell growth activators, including coenzyme 1. The addition of these components activates the growth of NK cells and promotes their expansion. This is because coenzyme 1 promotes the smooth progress of certain key metabolic pathways, providing the necessary energy and intermediates for cell growth and division, thereby promoting an increase in cell number and activating growth.

[0025] The method of the present invention can rapidly and massively expand NK cells while ensuring a high activity rate of NK cells, thereby obtaining NK cells with high purity and high killing activity. DETAILED DESCRIPTION

[0026] The present invention provides a method for improving the killing activity of NK cells, comprising the following steps:

[0027] (1) Separate peripheral blood to obtain PBMC cells;

[0028] (2) separating the PBMC cells to obtain NK cells;

[0029] (3) inoculating NK cells into NK cell culture medium for culture;

[0030] The NK cell culture medium comprises: 1-2 g / L L-lysine, 0.2-0.6 g / L methionine, 1-2 g / L L-tryptophan, 1-2 g / L L-proline, 6-10 g / L glutathione, 5-9 g / L coenzyme 1, 1-2 g / L silk peptide powder, 60-70 mL / L human plasma, 8-12 g / L human serum albumin, 0.16-0.18 μL / mL interleukin-10, 0.2-0.4 μL / mL interleukin-13, 0.3-0.5 μL / mL interleukin-18, and the rest is basal culture medium;

[0031] Preferably, the composition comprises 1.5 g / L L-lysine, 0.3-0.5 g / L methionine, 1.5 g / L L-tryptophan, 1.5 g / L L-proline, 7-9 g / L glutathione, 6-8 g / L coenzyme 1, 1.5 g / L silk peptide powder, 62-68 mL / L human plasma, 9-11 g / L human serum albumin, 0.17 μL / mL interleukin-10, 0.3 μL / mL interleukin-13, 0.4 μL / mL interleukin-18, and the rest as basal culture medium; The first step preferably comprises 1.5 g / L L-lysine, 0.4 g / L methionine, 1.5 g / L L-tryptophan, 1.5 g / L L-proline, 8 g / L glutathione, 7 g / L coenzyme 1, 1.5 g / L silk peptide powder, 64-66 mL / L human plasma, 10 g / L human serum albumin, 0.17 μL / mL interleukin-10, 0.3 μL / mL interleukin-13, 0.4 μL / mL interleukin-18, and the rest as basal culture medium; more preferably 1.5 g / L L-lysine, 0.4 g / L methionine, 1.5 g / L L-tryptophan, 1.5 g / L L-proline, 8 g / L glutathione, 7 g / L coenzyme 1, 1.5 g / L silk peptide powder, 65 mL / L human plasma, 10 g / L human serum albumin, 0.17 μL / mL interleukin-10, 0.3 μL / mL interleukin-13, 0.4 μL / mL interleukin-18, and the rest is basal culture medium.

[0032] In the present invention, the basal culture medium is Lonza X-VIVO 15 serum-free culture medium, SCGM serum-free culture medium or Takara NK cell serum-free culture medium; preferably Lonza X-VIVO 15 serum-free culture medium.

[0033] In the present invention, the culture temperature is 37-38° C., and the volume concentration of CO 2 is 5%-6%; preferably, the culture temperature is 37° C., and the volume concentration of CO 2 is 5%.

[0034] In the present invention, the culture time is 13 to 15 days, preferably 14 days.

[0035] In the present invention, the inoculated cell density is 7 to 9×10 5 / mL; preferably 8×10 5 pieces / mL.

[0036] In the present invention, during the culture process, the early rehydration medium is added every 2 to 3 days from the 0th to 7th day of culture, and the late rehydration medium is added every 2 to 3 days thereafter; preferably, the early rehydration medium is added every 3 days from the 0th to 7th day of culture, and the late rehydration medium is added every 3 days thereafter.

[0037] In the present invention, during the culture process, the cell density is always maintained at 7 to 9 × 10 5 / mL; preferably 8×10 5 pieces / mL.

[0038] In the present invention, the early rehydration culture medium comprises: 60-70 mL / L human plasma, 0.16-0.18 μL / mL interleukin-10, 0.2-0.4 μL / mL interleukin-13 and 0.3-0.5 μL / mL interleukin-18, and the rest is basal culture medium; preferably, 62-68 mL / L human plasma, 0.17 μL / mL interleukin-10, 0.3 μL / mL interleukin-13 and 0.4 μL / mL interleukin-18, and the rest is basal culture medium; further preferably, 65 mL / L human plasma, 0.17 μL / mL interleukin-10, 0.3 μL / mL interleukin-13 and 0.4 μL / mL interleukin-18, and the rest is basal culture medium.

[0039] In the present invention, the late rehydration medium comprises: 0.2-0.3 μL / mL interleukin-10, 0.5-0.6 μL / mL interleukin-13 and 0.6-0.7 μL / mL interleukin-18, and the rest is basal culture medium; preferably 0.25 μL / mL interleukin-10, 0.55 μL / mL interleukin-13 and 0.65 μL / mL interleukin-18, and the rest is basal culture medium.

[0040] The present invention also provides the use of NK cells prepared by the method for improving the killing activity of NK cells in the preparation of anti-tumor drugs.

[0041] In the present invention, the tumor is non-small cell lung cancer, chronic myeloid leukemia, colorectal cancer or breast cancer; preferably chronic myeloid leukemia.

[0042] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] A method for improving NK cell killing activity, comprising the following steps:

[0045] (1) collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0046] (2) Isolate NK cells from PBMCs using an NK cell isolation kit;

[0047] (3) The obtained NK cells were inoculated into NK cell culture medium and cultured for 13 days; the culture temperature was 38°C, the volume concentration of CO2 was 6%; the inoculated cell density was 9×10 5 pieces / mL.

[0048] (4) During the first 0-7 days of culture, the early rehydration medium was added every 2 days, and the late rehydration medium was added every 2 days thereafter; the cell density was always maintained at 9×10 5 pieces / mL.

[0049] The initial rehydration medium is composed of 60 mL / L human plasma, 0.16 μL / mL interleukin-10, 0.2 μL / mL interleukin-13, and 0.3 μL / mL interleukin-18, and the rest is Takara NK cell serum-free medium.

[0050] The late rehydration medium contains: 0.2 μL / mL interleukin-10, 0.5 μL / mL interleukin-13 and 0.6 μL / mL interleukin-18, and the rest is Takara NK cell serum-free medium.

[0051] The NK cell culture medium contains: 1 g / L L-lysine, 0.2 g / L methionine, 1 g / L L-tryptophan, 1 g / L L-proline, 6 g / L glutathione, 5 g / L coenzyme 1, 1 g / L silk peptide powder, 60 mL / L human plasma, 8 g / L human serum albumin, 0.16 μL / mL interleukin-10, 0.2 μL / mL interleukin-13, and 0.3 μL / mL interleukin-18, and the rest is Takara NK cell serum-free culture medium.

[0052] Example 2

[0053] A method for improving NK cell killing activity, comprising the following steps:

[0054] (1) collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0055] (2) Isolate NK cells from PBMCs using an NK cell isolation kit;

[0056] (3) The obtained NK cells were inoculated into NK cell culture medium and cultured for 15 days; the culture temperature was 37.5°C, the volume concentration of CO2 was 5.5%; the inoculated cell density was 7×10 5 pieces / mL.

[0057] (4) During the first 0 to 7 days of culture, the early rehydration medium was added every 3 days, and the late rehydration medium was added every 3 days thereafter; the cell density was always maintained at 7×10 5 pieces / mL.

[0058] The initial rehydration medium comprises: 70 mL / L human plasma, 0.18 μL / mL interleukin-10, 0.4 μL / mL interleukin-13 and 0.5 μL / mL interleukin-18, and the rest is SCGM serum-free medium.

[0059] The late rehydration medium contains: 0.3 μL / mL interleukin-10, 0.6 μL / mL interleukin-13 and 0.7 μL / mL interleukin-18, and the rest is SCGM serum-free medium.

[0060] The NK cell culture medium contains: 2 g / L L-lysine, 0.6 g / L methionine, 2 g / L L-tryptophan, 2 g / L L-proline, 10 g / L glutathione, 9 g / L coenzyme 1, 2 g / L silk peptide powder, 70 mL / L human plasma, 12 g / L human serum albumin, 0.18 μL / mL interleukin-10, 0.4 μL / mL interleukin-13, and 0.5 μL / mL interleukin-18, and the rest is SCGM serum-free medium.

[0061] Example 3

[0062] A method for improving NK cell killing activity, comprising the following steps:

[0063] (1) collecting peripheral blood, separating and collecting human peripheral blood mononuclear cells using lymphocyte separation solution, and washing to obtain human peripheral blood mononuclear cells;

[0064] (2) Isolate NK cells from PBMCs using an NK cell isolation kit;

[0065] (3) The obtained NK cells were inoculated into NK cell culture medium and cultured for 14 days; the culture temperature was 37°C, the volume concentration of CO2 was 5%; the inoculated cell density was 8×10 5 pieces / mL.

[0066] (4) During the first 0 to 7 days of culture, the early rehydration medium was added every 3 days, and the late rehydration medium was added every 3 days thereafter; the cell density was always maintained at 8×10 5 pieces / mL.

[0067] The initial rehydration medium comprises: 65 mL / L human plasma, 0.17 μL / mL interleukin-10, 0.3 μL / mL interleukin-13 and 0.4 μL / mL interleukin-18, and the rest is Lonza X-VIVO 15 serum-free medium.

[0068] The late rehydration medium contains: 0.25 μL / mL interleukin-10, 0.55 μL / mL interleukin-13 and 0.65 μL / mL interleukin-18, and the rest is Lonza X-VIVO 15 serum-free medium.

[0069] The NK cell culture medium contains: 1.5 g / L L-lysine, 0.4 g / L methionine, 1.5 g / L L-tryptophan, 1.5 g / L L-proline, 8 g / L glutathione, 7 g / L coenzyme 1, 1.5 g / L silk peptide powder, 65 mL / L human plasma, 10 g / L human serum albumin, 0.17 μL / mL interleukin-10, 0.3 μL / mL interleukin-13, and 0.4 μL / mL interleukin-18, and the rest is Lonza X-VIVO 15 serum-free medium.

[0070] In Comparative Example 1, the other methods were the same as those in Example 3, except that interleukin-10, interleukin-13, and interleukin-18 in the NK cell culture medium were replaced with equal amounts of interleukin-15, interleukin-18, and interleukin-21.

[0071] Comparative Example 2: Other methods were the same as in Example 3, except that interleukin-10, interleukin-13, and interleukin-18 in the early rehydration medium and the late rehydration medium were replaced with equal amounts of interleukin-15, interleukin-18, and interleukin-21.

[0072] Comparative Example 3: Other methods were the same as those in Example 3, except that L-lysine, methionine, L-tryptophan, and L-proline were not added to the NK cell culture medium.

[0073] Comparative Example 4: Other methods were the same as those in Example 3, except that glutathione and silk peptide powder were not added to the NK cell culture medium.

[0074] Experimental Example 1

[0075] The number of NK cells during the culture process of Example 3 and Comparative Examples 1 to Comparative Examples 4 was counted, and the expansion multiples were calculated. Two parallel experiments were performed in each group, and the results were averaged, as shown in Table 1.

[0076] Table 1 Cell expansion times

[0077] Group No. 0d 3d 7d 11d 14th day Example 3 1 27.6 68.5 124.3 211.6 Comparative Example 1 1 25.5 59.3 112.4 201.3 Comparative Example 2 1 25.1 57.6 108.5 195.7 Comparative Example 3 1 23.2 49.8 101.2 183.4 Comparative Example 4 1 24.3 58.7 96.4 178.2

[0078] As shown in Table 1, the culture method provided by the present invention can expand the cells by more than 200 times after culturing for 2 weeks, indicating that the method of the present invention can effectively promote the expansion of NK cells.

[0079] Experimental Example 2

[0080] Take the NK cells cultured in Example 3 and Comparative Examples 1 to 4, and resuspend the cells in RPMI1640 medium to a concentration of 1×10 6 The cell suspension of 100 cells / mL is ready for use;

[0081] K562 (human chronic myeloid leukemia) cells were used as target cells and resuspended in RPMI1640 medium containing 2% FBS to a volume of 1×10 5 The cell suspension of 100 cells / mL is ready for use;

[0082] In each experimental group, cells were inoculated into 96-well plates at effector-target ratios of 1:1, 5:1, and 10:1. Effector cell wells and target cell wells were set, and 5 replicate wells were inoculated for each culture method. The plates were cultured at 37°C and 5% CO2 for 24 h. The absorbance was measured using the MTT method, and the killing rate was calculated. The results were averaged and shown in Table 2 below.

[0083] Table 2 Killing rate

[0084] Group 1：1 5：1 10：1 Example 3 29.54％ 75.38％ 88.14％ Comparative Example 1 24.33％ 68.52％ 82.62％ Comparative Example 2 22.34％ 65.26％ 80.75％ Comparative Example 3 27.52％ 71.14％ 84.34％ Comparative Example 4 26.34％ 70.29％ 83.06％

[0085] As shown in Table 2, the NK cells cultured in the present invention have an excellent killing effect, which can reach up to 88.14%, which is significantly better than other comparative examples.

[0086] From the data obtained from Comparative Examples 1 to 4, it can be seen that the selection of interleukin types is very important, and its effect on the NK cell killing effect is the most significant, and the addition of amino acids, glutathione and silk peptide powder has the most significant effect on the expansion of NK cells. Therefore, in the process of NK cell culture, it is necessary to select the appropriate interleukin type, otherwise it will affect the killing effect of NK cells, and only when the complete component selection of the present invention is used can the best culture effect be achieved.

[0087] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for improving the killing activity of NK cells, characterized in that: The steps include: (1) Separate peripheral blood to obtain PBMC cells; (2) separating the PBMC cells to obtain NK cells; (3) inoculating NK cells into NK cell culture medium for culture; The NK cell culture medium comprises: 1-2 g / L L-lysine, 0.2-0.6 g / L methionine, 1-2 g / L L-tryptophan, 1-2 g / L L-proline, 6-10 g / L glutathione, 5-9 g / L coenzyme 1, 1-2 g / L silk peptide powder, 60-70 mL / L human plasma, 8-12 g / L human serum albumin, 0.16-0.18 μL / mL interleukin-10, 0.2-0.4 μL / mL interleukin-13, 0.3-0.5 μL / mL interleukin-18, and the rest is a basic culture medium.

2. A method for improving the killing activity of NK cells according to claim 1, characterized in that: The basic culture medium is Lonza X-VIVO 15 serum-free culture medium, SCGM serum-free culture medium or Takara NK cell serum-free culture medium.

3. A method for improving the killing activity of NK cells according to claim 1, characterized in that: The culture temperature is 37-38° C., and the volume concentration of CO 2 is 5%-6%.

4. A method for improving the killing activity of NK cells according to claim 1, characterized in that: The culture time is 13 to 15 days.

5. The method for improving the killing activity of NK cells according to claim 1, characterized in that: The inoculated cell density is 7-9×10 5 Pieces / mL.

6. The method for improving the killing activity of NK cells according to claim 1, characterized in that: During the culture process, the early rehydration medium was added every 2 to 3 days from the 0th to the 7th day of culture, and the late rehydration medium was added every 2 to 3 days thereafter; after the rehydration, the cell density was always maintained at 7 to 9×10 5 / mL; the early replenishment medium includes: 60~70mL / L human plasma, 0.16~0.18μL / mL interleukin-10, 0.2~0.4μL / mL interleukin-13 and 0.3~0.5μL / mL interleukin-18, and the rest is the basic culture medium; the late replenishment medium includes: 0.2~0.3μL / mL interleukin-10, 0.5~0.6μL / mL interleukin-13 and 0.6~0.7μL / mL interleukin-18, and the rest is the basic culture medium.