Culture method and application of high-secretion IFN-gamma CD4 + T cell

Abstract

The invention discloses a culture method and application of high-secretion IFN-gamma CD4 + T cells, and relates to the technical field of immune cell culture. The cell culture medium disclosed by the invention is prepared from the following components: IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, CD28, an STING agonist, astragaloside, ginsenoside Rh2 and astragalus polysaccharide. The CD4 + T cells cultured by the culture medium disclosed by the invention are cultured in vitro for 15 days, the content of the CD4 + T cells is greater than 70%, and the CD4 + T cells secrete IFN-gamma at a higher level compared with the CD4 + T cells cultured by a common CD4 + T cell culture medium, and in addition, the in-vitro killing rate of the CD4 + T cells to HCT116 intestinal cancer cells reaches 95% or above due to the added STING agonist, astragaloside, ginsenoside Rh2 and astragalus polysaccharide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immune cell culture, in particular to a culture method of CD4+ T cells with high secretion of IFN-γ and application thereof. BACKGROUND

[0002] IFN-γ is a key cytokine that plays an important role in immune regulation, antiviral response and antitumor immunity. It can activate macrophages and enhance their phagocytosis and killing of pathogens. In terms of anti-tumor, IFN-γ can play a role by inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and enhancing the killing effect of immune cells on tumor cells. CD4+ T cells are an important cell population in the immune system. Studies have shown that CD4+ T cells play an important role in the production of IFN-γ by CD8+ T cells. Therefore, obtaining CD4+ T cells with high secretion of IFN-γ is of great significance for in-depth study of its role in immune response.

[0003] There is a potential demand for CD4+ T cells with high secretion of IFN-γ in terms of tumor treatment, infectious disease treatment, and autoimmune function conditions. Recently, Japanese scholars Hashimoto et al. used single-cell RNA analysis technology to find that in super-elderly people (>110 years old), CD4+ T cells with high secretion of IFN-γ increased significantly, accounting for more than 80% of total T cells, while in the young group, the proportion was only 10%-20%. These cells have strong cytotoxicity and can directly kill infected cells or tumor cells, and can activate macrophages to enhance phagocytosis, thereby enhancing the immune surveillance ability of the body. Therefore, developing a culture method of CD4+ T cells with high secretion of IFN-γ can provide a new method for disease prevention and anti-aging.

[0004] At present, although there are some methods for T cell culture, there are problems such as high cost (sorting and use of special separation reagents) or inability to obtain CD4+ T cells with high secretion of IFN-γ in selective culture of CD4+ T cells. The establishment of this method can meet the needs of basic research and clinical application. SUMMARY

[0005] In view of the above defects of the prior art, the present application provides a culture method of CD4+ T cells with high secretion of IFN-γ and application thereof to solve the problems raised in the background art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The application discloses a culture method of CD4+ T cells with high IFN-gamma secretion, and belongs to the field of biomedicine. Step 3: inoculating the peripheral blood mononuclear cells into the initial culture bottle coated with the culture medium and adding the culture medium for culture; step 4: replacing the culture container based on the culture progress and supplementing the culture medium in the replaced culture container for continuous culture until a predetermined culture time is reached; Step 5: replacing the culture container based on the culture progress and supplementing the culture medium for continuous culture until a predetermined time is reached; step 6: collecting the cultured CD4+ T cells and performing related tests of the CD4+ T cells, wherein the related tests include purity, killing activity and secreted IFN-gamma detection.

[0007] As a further preferred scheme of the culture method of CD4+ T cells with high IFN-gamma secretion, in step 1, the IL-2 concentration is 100-1000 U / mL, the IL-7 concentration is 5-50 ng / mL, the IL-15 concentration is 10-50 ng / mL, the IL-18 concentration is 10-50 ng / mL; the AB plasma concentration is 2%-10%, the ConA concentration is 5-50 ug / mL, the OKT3 concentration is 0.5-50 ng / mL, the CD28 concentration is 0.5-50 ng / mL; the STING agonist concentration is 5-50 uM, the astragaloside concentration is 10-100 ug / mL, the ginsenoside Rh2 concentration is 10-100 ug / mL, and the astragalus polysaccharide concentration is 10-50 ug / mL.

[0008] As a further preferred scheme of the culture method of CD4+ T cells with high IFN-gamma secretion, the initial culture bottle is a T25 culture bottle, the culture container is replaced in time based on the culture progress, and the activated culture medium is supplemented in the replaced culture container in time and in an appropriate amount for continuous culture until a predetermined culture time is reached. After the peripheral blood mononuclear cells are cultured in the T75 culture bottle for 5 days, the peripheral blood mononuclear cells are transferred into a T225 culture bottle and the culture medium is supplemented in time and in an appropriate amount for continuous culture; After the peripheral blood mononuclear cells are continuously cultured in the T225 culture bottle for 4 days, the peripheral blood mononuclear cells are transferred into a 2.5L culture bag and the culture medium is supplemented in time and in an appropriate amount for continuous culture; After the peripheral blood mononuclear cells are continuously cultured in the culture bag for 6 days, the cultured CD4+ T cells are obtained.

[0009] As a further preferred scheme of the culture method of the CD4+ T cell secreting high IFN-γ of the present application, in step 2, the peripheral blood mononuclear cells are isolated from the human peripheral blood by density gradient centrifugation.

[0010] As a further preferred scheme of the culture method of the CD4+ T cell secreting high IFN-γ of the present application, the inoculation density of the peripheral blood mononuclear cells inoculated into the initial culture bottle is 2×10 6 individuals / mL.

[0011] As a further preferred scheme of the culture method of the CD4+ T cell secreting high IFN-γ of the present application, the inoculation of the peripheral blood mononuclear cells into the initial culture bottle and the addition of the appropriate amount of culture medium for culture, and the timely replacement of the culture container based on the culture progress and the timely and appropriate replenishment of the culture medium in the replaced culture container for continuous culture until the predetermined culture time are all carried out at a culture temperature of 37℃ and a carbon dioxide concentration of 5%.

[0012] As a further preferred scheme of the culture method of the CD4+ T cell secreting high IFN-γ of the present application, the criterion for the timely replacement of the culture container based on the culture progress and the timely and appropriate replenishment of the culture medium in the replaced culture container for continuous culture is to maintain the CD4+ T cell concentration in the culture container at 2×10 5 ~ 5×10 5 individuals / mL.

[0013] As a further preferred scheme of the culture method of the CD4+ T cell secreting high IFN-γ of the present application, the content of the CD4+ T cells collected from the cultured CD4+ T cells is >70%.

[0014] The CD4+ T cells cultured by the culture method of the CD4+ T cell secreting high IFN-γ have a killing effect on colorectal cancer cells.

[0015] The technical scheme provided by the embodiments of the present application can include the following beneficial effects: The application discloses a culture method and application of CD4+ T cells with high IFN-gamma secretion, and the culture medium comprises IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, CD28, a STING agonist, astragaloside, panax notoginseng saponin Rh2 and astragalus polysaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Results of ELISA detection of cytokine INF-gamma concentration in supernatants of cells in examples and comparative examples of the application are shown in the following table: Figure 2 Cell morphological pictures of peripheral blood mononuclear cells (PBMC) cultured for 0 days, 4 days, 8 days, 12 days and 15 days in Example 1 of the application are shown in the following table: Figure 3 Results of flow detection of purity of CD4+ T cells cultured for 15 days in Example 1 of the application are shown in the following table: Figure 4 Results of killing activity of CD4+ T cells obtained in examples and comparative examples of the application on HCT116 colorectal cancer cells are shown in the following table. DETAILED DESCRIPTION

[0017] The technical solutions in the examples of the application will be described clearly and in detail below with reference to the accompanying drawings in the examples of the application. The described examples are only some of the examples of the application. The technical solutions of the application will be further described in detail below with reference to specific embodiments.

[0018] The application provides a culture medium, which comprises IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, CD28, a STING agonist, astragaloside, panax notoginseng saponin Rh2 and astragalus polysaccharide.

[0019] The concentration of IL-2 in the medium is 100-1000 U / mL (for example, 100 U / mL, 200 U / mL, 300 U / mL, 450 U / mL, 600 U / mL, 800 U / mL, 1000 U / mL), the concentration of IL-7 is 5-50 ng / mL (for example, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL), the concentration of IL-15 is 10-50 ng / mL (for example, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL), the concentration of IL-18 is 10-50 ng / mL (for example, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL), the concentration of ConA is 5-50 μg / mL (for example, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL), the concentration of OKT3 is 0.5-50 ng / mL (for example, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL), the concentration of CD28 is 0.5-50 ng / mL (for example, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL), the concentration of STING agonist is 5-50 μM (for example, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM), the concentration of astragalus membranaceus glycoside is 10-100 μg / mL (for example, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL), the concentration of ginsenoside Rh2 is 10-100 μg / mL (for example, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL), the concentration of astragalus polysaccharide is 10-50 μg / mL (for example, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL), the concentration of AB plasma is 2%-10% (for example, 2%, 4%, 6%, 8%, 10%), and it should be noted that the concentration of AB plasma is 2%-10% means that the volume of AB plasma accounts for 2%-10% of the total volume of the medium.

[0020] The second aspect of the present application provides a culture method of CD4+ T cells with high secretion of IFN-γ, comprising: isolating peripheral blood mononuclear cells from human peripheral blood; Said peripheral blood mononuclear cells are inoculated into said initial culture flask and cultured with an appropriate amount of culture medium, which comprises IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, CD28, STING agonist, astragaloside IV, panax notoginseng saponin Rh2, and astragalus polysaccharide; The culture container is timely replaced based on the culture progress, and the culture medium is timely and appropriately supplemented in the replaced culture container for continuous culture until a predetermined culture time is reached. The cultured CD4+T cells are collected, and relevant tests of said CD4+T cells are performed, including purity, killing activity, and secreted IFN-γ detection.

[0021] Said initial culture flask is a T75 culture flask, and said timely replacement of the culture container based on the culture progress and the continuous culture of the culture medium in the replaced culture container until a predetermined culture time is reached comprise: After 5 days of culture in said T75 culture flask, said peripheral blood mononuclear cells are transferred to a T225 culture flask and continuously cultured with timely and appropriate supplementation of the culture medium; After 4 days of continuous culture in said T225 culture flask, said peripheral blood mononuclear cells are transferred to a 2.5L culture bag and continuously cultured with timely and appropriate supplementation of the culture medium; After 6 days of continuous culture in said culture bag, cultured CD4+T cells are obtained.

[0022] Said peripheral blood mononuclear cells are isolated from human peripheral blood by density gradient centrifugation.

[0023] Said peripheral blood mononuclear cells are inoculated into said initial culture flask at a density of 2×106. Said inoculation of said peripheral blood mononuclear cells into said initial culture flask and the culture with an appropriate amount of culture medium, and said timely replacement of the culture container based on the culture progress and the continuous culture of the culture medium in the replaced culture container until a predetermined culture time are all performed at a culture temperature of 37℃ and a carbon dioxide concentration of 5%.

[0024] Said timely replacement of the culture container based on the culture progress and the continuous culture of the culture medium in the replaced culture container until a predetermined culture time are performed according to a criterion that the concentration of CD4+T cells in the culture container is maintained at 5×105-2×106 / mL.

[0025] The content of CD4+T cells in said cultured CD4+T cells is >70%.

[0026] The third aspect of the present invention provides the application of CD4+ T cells cultured using the method described in any one of the above-mentioned methods for culturing CD4+ T cells that secrete high levels of IFN-γ in the treatment of colorectal cancer.

[0027] To better illustrate the technical effects of the present invention, the present invention has constructed the following embodiments and comparative examples for testing.

[0028] Example 1: The culture medium included IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, CD28, STING agonist, astragaloside A, ginsenoside Rh2, and astragalus polysaccharide. The concentrations of IL-2, IL-7, IL-15, IL-18, AB plasma, and astragalus polysaccharide were as follows: IL-2: 400 U / mL; IL-7: 5 ng / mL; IL-15: 10 ng / mL; IL-18: 20 ng / mL; AB plasma: 2%; ConA: 5 μg / mL; OKT3: 3 ng / mL; CD28: 5 ng / mL; STING agonist: 10 μM; astragaloside A: 50 μg / mL; ginsenoside Rh2: 50 μg / mL; and astragalus polysaccharide: 20 μg / mL.

[0029] The following method was used to culture CD4+ T cells: Human peripheral blood mononuclear cells (PBMCs) were isolated; PBMCs were seeded into T75 culture flasks and cultured with an appropriate amount of culture medium, with a PBMC seeding concentration of 2.0 × 10⁶ cells / mL; after 5 days of culture, the mononuclear cells were transferred to T225 culture flasks, and an appropriate amount of culture medium was added for continued culture, with a PBMC concentration of 2.5 × 10⁶ cells / mL; after 9 days of culture, the mononuclear cells were transferred to 2.5L culture bags, and an appropriate amount of culture medium was added for continued culture, with a PBMC concentration of 2.5 × 10⁶ cells / mL; after 15 days of culture, the cells were collected for purity, cytotoxic activity, and cytokine release assays.

[0030] Comparative Example 1: The culture medium included IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, and CD28. The concentrations of IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, and CD28 were 400 U / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 2% and 5 μg / mL, respectively. The culture medium did not contain STING agonist, astragaloside A, ginsenoside Rh2, or astragalus polysaccharide.

[0031] The following method was used to culture CD4+ T cells: Human peripheral blood mononuclear cells (PBMCs) were isolated; PBMCs were seeded into T75 culture flasks and cultured with an appropriate amount of culture medium, with a PBMC seeding concentration of 2.0 × 10⁶ cells / mL; after 5 days of culture, the mononuclear cells were transferred to T225 culture flasks, and an appropriate amount of culture medium was added for continued culture, with a PBMC concentration of 2.5 × 10⁶ cells / mL; after 9 days of culture, the mononuclear cells were transferred to 2.5L culture bags, and an appropriate amount of culture medium was added for continued culture, with a PBMC concentration of 2.5 × 10⁶ cells / mL; after 15 days of culture, the cells were collected for purity, cytotoxic activity, and cytokine release assays.

[0032] Comparative Example 2: A commercially available CD4+ T cell culture medium, including basal medium, fetal bovine serum, cell growth factors, and penicillin / streptomycin solution; the specific reagent formulation is unknown.

[0033] Test Results: The concentration of IFN-γ in the collected CD4+ T cell supernatant was detected using ELISA. The results are as follows: Figure 1 As shown in Table 1.

[0034] Table 1 shows the IFN-γ secretion data of CD4+ T cells in each embodiment and comparative example.

[0035] Table 1 Cell morphology was photographed and preserved on days 0, 4, 8, 12, and 15 of culture using an inverted microscope. Figure 2 As shown. The content of CD4⁺ T cells (CD3+CD4+) in the collected cells was detected by flow cytometry, and the result was 70.6%, as indicated. Figure 3 As shown.

[0036] The killing activity of collected CD4+ T cells against HCT116 colorectal cancer cells was detected using an LDH release assay. The specific method is as follows: HCT116 colorectal cancer cells (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were used as target cells, and CD4+ T cells were used as effector cells. HCT116 colorectal cancer cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension. The cells were counted by trypan blue staining, and the cell density was adjusted to 1×10⁵ cells / mL. 50 μL of the HCT116 cell suspension was added to each well of a 96-well plate, and effector cells (CD4+ T cells) were added at different effector / target ratios (1:1, 5:1, 10:1). Cells), 50 μL per well, with separate wells for effector cells and target cells spontaneous release, target cell maximum release, and culture medium spontaneous release, and a volume correction control well, 100 μL per well, all in triplicate; incubated at 37°C, 5% CO2 for 12 h; 45 min before the end of the reaction, 10 μL of lysis buffer was added to each target cell maximum release well; after the reaction, 50 μL of LDH enzyme reaction solution and 50 μL of supernatant were transferred from each well to a new 96-well plate, and reacted at room temperature in the dark for 30 min, followed by 50 μL of reaction stop solution. The OD value was measured using a microplate reader; the killing activity was calculated using the following formula: Killing activity (%) = (OD value of test tube - OD value of target cell spontaneous release tube - OD value of effector cell spontaneous release tube) / (OD value of target cell maximum release tube - OD value of target cell spontaneous release tube) × 100%. The calculation results are shown below. Figure 4 As shown in Table 2, the kill rate of Example 1 reached 97%.

[0037] Table 2 shows the CD4+ T cell expansion folds of each example and comparative example under different effect-to-target ratios.

[0038] Table 2 Based on the culture medium and method of this invention, co-culturing human peripheral blood mononuclear cells in vitro for 2 weeks resulted in a CD4+ T cell ratio >70% and enhanced CD4+ T cell killing activity, achieving a killing rate of >95% against HCT116 colorectal cancer cells (effect-to-target ratio of 10:1). Furthermore, the CD4+ T cells obtained through this culture method secreted higher levels of IFN-γ (compared to commercially available CD4+ T cell culture medium). Example 1 showed an IFN-γ concentration of 602.75 ng / mL in the CD4+ T cell supernatant, while Comparative Example 2 (commercially available CD4+ T cell culture medium) showed an IFN-γ concentration of 5.87 ng / mL. Moreover, under an effect-to-target ratio of 10:1, the addition of STING agonist, astragaloside A, ginsenoside Rh2, and astragalus polysaccharide resulted in an in vitro killing rate of over 95% for HCT116 colorectal cancer cells by CD4+ T cells.

[0039] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0040] It will be apparent to those skilled in the art that this application is not limited to the details of the exemplary embodiments described above, and that this application can be implemented in other specific forms without departing from the spirit or essential characteristics of this application. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of this application is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within this application. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for culturing CD4+ T cells that highly secrete IFN-γ, characterized in that, include: Step 1: Obtain a culture medium containing IL-2, IL-7, IL-15, IL-18, AB plasma, ConA, OKT3, CD28, STING agonist, astragaloside A, ginsenoside Rh2, and astragalus polysaccharide; Step 2: Isolate peripheral blood mononuclear cells from human peripheral blood; Step 3: Seed the peripheral blood mononuclear cells into the coated initial culture flask and add the culture medium for culture; Step 4: Change the culture container according to the culture progress, and add the culture medium to the new culture container to continue culture until the predetermined culture time is reached; Step 5: Replace the culture container and replenish the culture medium according to the culture progress, and continue culturing until the predetermined time; Step 6: Collect the cultured CD4+ T cells and perform relevant tests on the CD4+ T cells, including purity, cytotoxic activity and secreted IFN-γ detection.

2. The method for culturing CD4+ T cells that secrete high levels of IFN-γ according to claim 1, characterized in that, In step 1, the concentrations of IL-2, IL-7, IL-15, and IL-18 were 10–50 ng / mL; the plasma concentrations of AB were 2%–10%; the concentrations of ConA, OKT3, and CD28 were 0.5–50 ng / mL; the concentrations of STING agonist, astragaloside A, ginsenoside Rh2, and astragalus polysaccharide were 10–50 μg / mL.

3. The method for culturing CD4+ T cells that secrete high levels of IFN-γ according to claim 1, characterized in that, The initial culture flask is a T25 culture flask. The process of changing the culture container as needed based on the culture progress, and adding the activation culture medium to the new culture container as needed to continue culturing until the predetermined culture time is reached includes: After culturing in the T75 culture flask for 5 days, the peripheral blood mononuclear cells were transferred to the T225 culture flask and the culture medium was added as needed to continue culturing. After culturing in the T225 culture flask for another 4 days, the peripheral blood mononuclear cells were transferred to a 2.5L culture bag and the culture medium was added as needed to continue culturing. After culturing in the culture bag for another 6 days, cultured CD4+ T cells were obtained.

4. A method for culturing CD4+ T cells that secrete high levels of IFN-γ according to claim 3. Its features are, In step 2, peripheral blood mononuclear cells are separated from human peripheral blood using density gradient centrifugation.

5. A method for culturing CD4+ T cells that secrete high levels of IFN-γ according to claim 3. Its features are, The seeding density of the peripheral blood mononuclear cells in the initial culture flask is 2 × 10⁻⁶. 6 per mL.

6. A method for culturing CD4+ T cells that secrete high levels of IFN-γ according to claim 3. Its features are, The process of seeding peripheral blood mononuclear cells into an initial culture flask, adding an appropriate amount of culture medium for culture, and changing the culture container in a timely manner based on the culture progress, and adding the appropriate amount of culture medium to the changed culture container to continue culturing until the predetermined culture time is reached, is carried out at a culture temperature of 37°C and a carbon dioxide concentration of 5%.

7. The method for culturing CD4+ T cells that secrete high levels of IFN-γ according to claim 6, characterized in that, The principle of timely replacement of the culture container based on the culture progress, and timely and appropriate replenishment of the culture medium in the replaced culture container to continue the culture, is to maintain the CD4⁺T cell concentration in the culture container at 2×10⁻⁶. 5 ~5×10 5 per mL.

8. The method for culturing CD4+ T cells that secrete high levels of IFN-γ according to claim 7, characterized in that, The collected cultured CD4+ T cells contained >70% CD4+ T cells.

9. The killing effect of CD4+ T cells cultured according to any one of claims 1 to 8 on colorectal cancer cells.

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