NKT cell, culture medium and culture method thereof

Abstract

The invention discloses an NKT cell, a culture medium and a culture method thereof, and relates to the technical field of cell culture, the NKT cell comprises a basal culture medium, a cell factor combination, a plant extract compound, a metabolism regulator and an antioxidant; wherein the plant extract compound is prepared from astragalus polysaccharide, ganoderic acid, tea polyphenol, asiaticoside and notoginsenoside R1, and the mass ratio of the astragalus polysaccharide to the ganoderic acid to the tea polyphenol to the asiaticoside to the notoginsenoside R1 is (3 to 5) to (1 to 2) to 1 to (0.5 to 1) to (0.5 to 1). According to the NKT cell, the culture medium and the culture method of the NKT cell, through the synergistic effect of the six-membered plant extract compound, the expression of cyclin is promoted, the G1 / S phase conversion is accelerated, and the cell amplification amount is remarkably increased; the ratio of plant extracts and high astragalus polysaccharide are dynamically adjusted, so that cell proliferation and induced differentiation of high ganoderic acid are promoted, cells are forced to turn to efficient-productivity oxidative phosphorylation metabolism in cooperation with 2-deoxyglucose, and the cell multiplication time is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to NKT cells, a culture medium and a culture method thereof. Background Art

[0002] NKT (Natural Killer T) cells, as a special immune cell subset that has both T cell receptors (TCR) and NK cell receptors, play a key role in anti-tumor immunity, regulation of autoimmune diseases and inhibition of transplant rejection. Studies have shown that CD3 + CD56 + Double-positive NKT cells can activate anti-tumor immunity by secreting cytokines such as IFN-γ and TNF-α, and directly kill tumor cells by expressing perforin and granzyme B. Their functional activity is significantly positively correlated with clinical treatment effects.

[0003] The Chinese patent publication number CN105219712A discloses a NKT cell culture medium and a NKT cell culture method. The NKT cell culture medium includes albumin, vitamin C, ginsenosides, lentinan, cytokines, and serum-free basal medium. It can effectively promote the induction and expansion of NKT cells and improve the toxicity of the cultured NKT cells. Experimental results show that when the culture medium provided by the invention is used for in vitro NKT cell culture, the NKT cell expansion amount and the CD3 + 、CD56 + The double-positive expression levels and the toxic effects of cultured NKT cells on K562 cells were far superior to those of traditional culture media, indicating that the NKT cell culture medium provided by the present invention is beneficial for the induction and expansion of NKT cells, can improve the toxic effects of cultured NKT cells, and thus improve the disease treatment effects of cultured NKT cells. However, the above-mentioned prior art has the following problems: serum-free culture media use a simple combination of plant extracts (such as lentinan and ginsenosides) and cytokines, lack multi-pathway regulation of NKT cell proliferation, differentiation, and metabolism, and fail to achieve synergistic optimization among the components. To address the above-mentioned problems, the present invention provides NKT cells, culture media, and culture methods thereof to address the aforementioned problems. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a NKT cell, a culture medium and a culture method thereof, which solve the problems mentioned in the above background technology.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: a NKT cell culture medium, comprising: a basal culture medium, a cytokine combination, a plant extract complex, a metabolic regulator, and an antioxidant;

[0006] The plant extract complex comprises astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1, and the mass ratio of astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1 is (3-5): (1-2): 1: (0.5-1): (0.5-1);

[0007] The metabolic regulator comprises sodium pyruvate, L-carnitine and 2-deoxyglucose, and the molar ratio of sodium pyruvate, L-carnitine and 2-deoxyglucose is (5-10): (2-5): (1-3);

[0008] The antioxidants include glutathione, lipoic acid and astaxanthin loaded on chitosan-polylactic acid glycolic acid nanoparticles, and the particle size of the nanoparticles is 100-200 nm;

[0009] The culture medium also includes 1-3 wt % of fucosylated bovine serum albumin.

[0010] Preferably, the basal culture medium is serum-free DMEM / F12 culture medium containing 10 mmol / L HEPES buffer and 0.1 mmol / L non-essential amino acids.

[0011] Preferably, the cytokine combination includes IL-2 1000-1200 IU / mL, IL-15 20-40 ng / mL, and IL-21 10-30 ng / mL.

[0012] Preferably, the astragalus polysaccharide is 50-100 μg / mL, ganoderic acid is 20-35 μg / mL, and tea polyphenols is 10-25 μg / mL.

[0013] Preferably, the content of glutathione is 5-10 μg / mL, the content of lipoic acid is 2-5 μg / mL, the content of astaxanthin is 1-2 μg / mL, and the nanoparticle encapsulation efficiency is ≥80%.

[0014] A method for culturing NKT cells in an NKT cell culture medium comprises the following culturing steps:

[0015] Step 1: Isolate and obtain mononuclear cells, resuspend them in NKT cell culture medium, and adjust the cell density to 0.8×10 6 -2×10 6 / mL;

[0016] Step 2: Inoculate the cell suspension into a culture container and culture at 37°C and 5% CO2, dynamically adjusting the culture medium composition and culture parameters during the culture process;

[0017] Step 3: After culturing for 8-10 days, collect the cells and screen to obtain NKT cells.

[0018] Preferably, during the culture period of 0-3 days, the mass ratio of the plant extract complex astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1 in the culture medium is (4-5): (1.5-2): 1:1:1;

[0019] After 4-7 days of culture, the mass ratio of the plant extract complex astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginseng saponin R1 is (3-4): (1-1.5): 1:0.5:0.5, and 2-deoxyglucose is added to 1-3 mmol / L.

[0020] Preferably, the concentration of L-carnitine in the metabolic regulator is 2 mmol / L during the culture period from day 0 to day 5 and 5 mmol / L starting from day 6, and antioxidants are supplemented at the same time.

[0021] Beneficial effects

[0022] The present invention provides a NKT cell, culture medium, and culture method thereof. Compared with the prior art, it has the following advantages:

[0023] (1) The NKT cells, culture medium and culture method thereof promote the expression of cell cycle proteins, accelerate the G1 / S phase transition and significantly increase the amount of cell expansion through the synergistic effect of a six-component plant extract complex (astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginseng saponin R1); by dynamically adjusting the proportion of plant extracts and high astragalus polysaccharide, cell proliferation is promoted, high ganoderic acid induces differentiation, and combined with 2-deoxyglucose, forces cells to switch to oxidative phosphorylation metabolism with high energy production, thereby shortening the cell doubling time.

[0024] (2) The NKT cells, culture medium and culture method thereof, through sodium pyruvate + L-carnitine + 2-deoxyglucose, synergistically regulate mitochondrial function, promote the expression of cytotoxicity-related genes (such as FasL, TRAIL), and increase the killing rate of K562 cells. At the same time, IL-21 and tea polyphenols synergistically enhance the maintenance of NKT cell memory phenotype, thereby improving the toxic effect during the secondary immune response; chitosan-polylactic acid glycolic acid nanoparticles are loaded with glutathione, lipoic acid and astaxanthin to form a fat-soluble-water-soluble antioxidant network, reduce the level of mitochondrial reactive oxygen species, and reduce the cell apoptosis rate. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0026] Example 1

[0027] A NKT cell culture medium and a method for culturing NKT cells, comprising the following culturing steps:

[0028] Step 1: Isolate and obtain mononuclear cells, resuspend them in NKT cell culture medium, and adjust the cell density to 0.8×10 6 / mL;

[0029] The NKT cell culture medium includes: serum-free DMEM / F12 medium containing 10 mmol / L HEPES buffer and 0.1 mmol / L non-essential amino acids, IL-2 1000 IU / mL, IL-15 20 ng / mL, and IL-21 10 ng / mL, a plant extract complex, a metabolic regulator, and an antioxidant, wherein the plant extract complex includes astragalus polysaccharide 50 μg / mL, ganoderic acid 20 μg / mL, tea polyphenols 10 μg / mL, asiaticoside 25 μg / mL, and notoginseng saponin R1L 30 μg / mL; the metabolic regulator includes sodium pyruvate, L-carnitine, and 2-deoxyglucose; the antioxidant includes glutathione 5 μg / mL, lipoic acid 2 μg / mL, and astaxanthin 1 μg / mL loaded on chitosan-polylactic-co-glycolic acid nanoparticles, wherein the nanoparticles have a particle size of 100 nm;

[0030] Step 2: Inoculate the cell suspension into a culture container and culture at 37°C and 5% CO2, dynamically adjusting the culture medium composition and culture parameters during the culture process;

[0031] Among them, during the 0-3 days of culture, the mass ratio of the plant extract complex, astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1 in the culture medium was 4:1.5:1:1:1;

[0032] During the 4-7 days of culture, the mass ratio of the plant extract complex, astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside, and notoginsenoside R1, was 3:1:1:0.5:0.5, 2-deoxyglucose was added to 1 mmol / L, and the concentration of L-carnitine in the metabolic regulator was 2 mmol / L during the 0-5 days of culture;

[0033] Step 3: After culturing for 8-10 days, the cells are collected and screened to obtain NKT cells;

[0034] Among them, the concentration of L-carnitine in the metabolic regulator was 5 mmol / L starting from the 6th day of culture, and antioxidants were supplemented at the same time.

[0035] Example 2

[0036] A NKT cell culture medium and a method for culturing NKT cells, comprising the following culturing steps:

[0037] Step 1: Isolate and obtain mononuclear cells, resuspend them in NKT cell culture medium, and adjust the cell density to 1.4×10 6 / mL;

[0038] The NKT cell culture medium includes: serum-free DMEM / F12 medium containing 10 mmol / L HEPES buffer and 0.1 mmol / L non-essential amino acids, IL-2 1100 IU / mL, IL-15 30 ng / mL, and IL-21 20 ng / mL, a plant extract complex, a metabolic regulator, and an antioxidant, wherein the plant extract complex includes astragalus polysaccharide 80 μg / mL, ganoderic acid 25 μg / mL, tea polyphenols 20 μg / mL, asiaticoside 30 μg / mL, and notoginseng saponin R1L 35 μg / mL; the metabolic regulator includes sodium pyruvate, L-carnitine, and 2-deoxyglucose; the antioxidant includes glutathione 8 μg / mL, lipoic acid 3 μg / mL, and astaxanthin 1.5 μg / mL loaded on chitosan-polylactic-co-glycolic acid nanoparticles, wherein the nanoparticles have a particle size of 150 nm;

[0039] Step 2: Inoculate the cell suspension into a culture container and culture at 37°C and 5% CO2, dynamically adjusting the culture medium composition and culture parameters during the culture process;

[0040] Among them, during the 0-3 days of culture, the mass ratio of the plant extract complex, astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1 in the culture medium was 4.5:1.8:1:1:1;

[0041] On days 4-7 of culture, the mass ratio of the plant extract complex, astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside, and notoginsenoside R1, was 3.5:1.2:1:0.5:0.5, 2-deoxyglucose was added to 2 mmol / L, and the concentration of L-carnitine in the metabolic regulator was 2 mmol / L on days 0-5 of culture;

[0042] Step 3: After culturing for 8-10 days, the cells are collected and screened to obtain NKT cells;

[0043] Among them, the concentration of L-carnitine in the metabolic regulator was 5 mmol / L starting from the 6th day of culture, and antioxidants were supplemented at the same time.

[0044] Example 3

[0045] A NKT cell culture medium and a method for culturing NKT cells, comprising the following culturing steps:

[0046] Step 1: Isolate and obtain mononuclear cells, resuspend in NKT cell culture medium, and adjust the cell density to 2×10 6 / mL;

[0047] The NKT cell culture medium includes: serum-free DMEM / F12 medium containing 10 mmol / L HEPES buffer and 0.1 mmol / L non-essential amino acids, 1200 IU / mL of IL-2, 40 ng / mL of IL-15, and 30 ng / mL of IL-21, a plant extract complex, a metabolic regulator, and an antioxidant, wherein the plant extract complex includes 100 μg / mL of astragalus polysaccharide, 35 μg / mL of ganoderic acid, 25 μg / mL of tea polyphenols, 40 μg / mL of asiaticoside, and 40 μg / mL of notoginseng saponin R1L; the metabolic regulator includes sodium pyruvate, L-carnitine, and 2-deoxyglucose; the antioxidant includes 10 μg / mL of glutathione, 5 μg / mL of lipoic acid, and 2 μg / mL of astaxanthin loaded on chitosan-polylactic-co-glycolic acid nanoparticles, wherein the nanoparticles have a particle size of 200 nm;

[0048] Step 2: Inoculate the cell suspension into a culture container and culture at 37°C and 5% CO2, dynamically adjusting the culture medium composition and culture parameters during the culture process;

[0049] Among them, during the culture period of 0-3 days, the mass ratio of the plant extract complex, astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1 in the culture medium was 5:2:1:1:1;

[0050] On days 4-7 of culture, the mass ratio of the plant extract complex, astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside, and notoginsenoside R1, was 4:1.5:1:0.5:0.5, 2-deoxyglucose was added to 3 mmol / L, and the concentration of L-carnitine in the metabolic regulator was 2 mmol / L on days 0-5 of culture;

[0051] Step 3: After culturing for 8-10 days, the cells are collected and screened to obtain NKT cells;

[0052] Among them, the concentration of L-carnitine in the metabolic regulator was 5 mmol / L starting from the 6th day of culture, and antioxidants were supplemented at the same time.

[0053] Comparative Example 1

[0054] Compared with Example 1, the difference is that the plant extract complex is not added; the rest is the same as Example 1.

[0055] Comparative Example 2

[0056] Compared with Example 1, the difference is that no metabolic regulator is added; the rest is the same as Example 1.

[0057] Comparative Example 3

[0058] Compared with Example 1, the difference is that no antioxidant is added; the rest is the same as Example 1.

[0059] The expansion times of cells after culture, the expression of CD3 in NKT cells and the expression of IL-6 in Examples 1 to 3 and Comparative Examples 1 to 3 were counted respectively. + CD56 + The cell percentage and cell apoptosis rate are shown in Table 1.

[0060] Table 1

[0061] Amplification factor <![CDATA[CD3 + CD56 + Ratio / %]]> Cell apoptosis rate / % Example 1 442 83.2 7.6 Example 2 436 81.9 7.9 Example 3 433 78.3 8.4 Comparative Example 1 406 72.9 10.1 Comparative Example 2 389 68.6 11.3 Comparative Example 3 375 65.1 11.9

[0062] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0063] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0064] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A NKT cell culture medium, characterized in that include: Basal culture media, cytokine combinations, plant extract complexes, metabolic regulators, antioxidants; The plant extract complex comprises astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1, and the mass ratio of astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1 is (3-5): (1-2): 1: (0.5-1): (0.5-1); The metabolic regulator comprises sodium pyruvate, L-carnitine and 2-deoxyglucose, and the molar ratio of sodium pyruvate, L-carnitine and 2-deoxyglucose is (5-10): (2-5): (1-3); The antioxidants include glutathione, lipoic acid and astaxanthin loaded on chitosan-polylactic acid glycolic acid nanoparticles, and the particle size of the nanoparticles is 100-200 nm; The culture medium also includes 1-3 wt % of fucosylated bovine serum albumin.

2. The NKT cell, culture medium and culture method according to claim 1, characterized in that: The basal culture medium is serum-free DMEM / F12 culture medium containing 10 mmol / L HEPES buffer and 0.1 mmol / L non-essential amino acids.

3. The NKT cell, culture medium and culture method thereof according to claim 1, characterized in that: The cytokine combination includes IL-2 1000-1200 IU / mL, IL-15 20-40 ng / mL, and IL-21 10-30 ng / mL.

4. The NKT cell, culture medium and culture method thereof according to claim 1, characterized in that: The astragalus polysaccharide is 50-100 μg / mL, ganoderic acid is 20-35 μg / mL, tea polyphenols is 10-25 μg / mL, asiaticoside is 25-40 μg / mL and notoginsenoside R1L is 30-40 μg / mL.

5. The NKT cells, culture medium and culture method thereof according to claim 1, characterized in that: The content of glutathione is 5-10 μg / mL, the content of lipoic acid is 2-5 μg / mL, the content of astaxanthin is 1-2 μg / mL, and the nanoparticle encapsulation efficiency is ≥80%.

6. A method for culturing NKT cells in the NKT cell culture medium according to claim 1, characterized in that: The following cultivation steps are included: Step 1: Isolate and obtain mononuclear cells, resuspend them in NKT cell culture medium, and adjust the cell density to 0.8×10 6 -2×10 6 / mL; Step 2: Inoculate the cell suspension into a culture container and culture at 37°C and 5% CO2, dynamically adjusting the culture medium composition and culture parameters during the culture process; Step 3: After culturing for 8-10 days, collect the cells and screen to obtain NKT cells.

7. The method for culturing NKT cells using a NKT cell culture medium according to claim 6, wherein: During the 0-3 days of culture, the mass ratio of the plant extract complex astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginsenoside R1 in the culture medium was (4-5): (1.5-2): 1:1:1; After 4-7 days of culture, the mass ratio of the plant extract complex astragalus polysaccharide, ganoderic acid, tea polyphenols, asiaticoside and notoginseng saponin R1 is (3-4): (1-1.5): 1:0.5:0.5, and 2-deoxyglucose is added to 1-3 mmol / L.

8. The method for culturing NKT cells using a NKT cell culture medium according to claim 6, wherein: The concentration of L-carnitine in the metabolic regulator is 2 mmol / L during the culture period from day 0 to day 5 and 5 mmol / L starting from day 6, and antioxidants are supplemented at the same time.