Γδt cell culture medium, culture method and use

Abstract

Provided in the present invention are a γδT cell culture medium, a culture method and the use. The γδT cell culture medium comprises a basal medium, IL-2, IL-7, IL-15, IL-18, IL-21, zoledronic acid and berberine at a concentration of 0.25-5 μM.

Description

Gamma delta T cell culture medium, culture method and application TECHNICAL FIELD

[0001] The present application relates to the technical field of gamma delta T cell culture, in particular to a gamma delta T cell culture medium, culture method and application. BACKGROUND

[0002] Gamma delta T cells are a kind of non-classical T cells in the immune system, mainly distributed in mucosa and subcutaneous tissue of intestinal tract, respiratory tract and urogenital tract, and are one of the main components of intraepidermal lymphocytes and mucosal tissue intraepithelial lymphocytes (IEL). In healthy adults, gamma delta T cells account for 1% to 5% of peripheral blood T cells. However, gamma delta T cells can directly kill cancer cells and tumor stem cells, recognize carcinoembryonic antigen, and can also directly kill tumor cells through perforin granule protease pathway and other ways. In addition, gamma delta T cells also play an important role in controlling viral infection and regulating autoimmune diseases.

[0003] In view of the significant therapeutic effect of gamma delta T cell therapy in cancer and viral diseases, it is an urgent problem to be solved to culture high-purity active gamma delta T cells in vitro. However, the existing culture method of gamma delta T cells has the problem of low expansion rate, and how to improve the expansion rate of gamma delta T cells is a problem to be solved. SUMMARY

[0004] The present application provides a gamma delta T cell culture medium, culture method and application, and the main purpose of the present application is to solve the problem of low expansion rate of gamma delta T cells in the existing culture technology.

[0005] The content of the present application is as follows:

[0006] The present application provides a gamma delta T cell culture medium, which comprises: a basic culture medium, a cytokine, a bisphosphonate and berberine, and the concentration of the berberine is 0.25-5 μM.

[0007] In the gamma delta T cell culture medium of the present application, the basic culture medium comprises one of AIM-V culture medium, KBM-581 culture medium and RPMI-1640, which provides basic nutrients and substances for cell proliferation, and is also the survival environment for cell growth and reproduction. In the present application, the selected basic culture medium is AIM-V culture medium.

[0008] In the culture medium, bisphosphonate, cytokine and berberine are also added, and the combination of each component in the culture medium also improves the expansion rate of gamma delta T cells.

[0009] Berberine, also known as Coptis, is a quaternary ammonium alkaloid isolated from traditional Chinese medicine Coptis, which is the main effective component of Coptis. Due to its antibacterial effect, berberine can be used as an antibacterial component for cell culture in some cases, so it can provide the survival rate of some cells. The inventors of the present application found that berberine as a component of the gamma delta T cell culture medium of the present application has a promoting effect on the proliferation of gamma delta T cells, which not only improves the survival rate, but also significantly improves the expansion multiple of gamma delta T cells.

[0010] In an alternative embodiment of the first aspect of the present application, the gamma delta T cell culture medium further comprises 5-10% autologous plasma.

[0011] The addition of autologous plasma can meet the nutritional needs of cell culture, and help the growth and proliferation of cells in the culture environment.

[0012] Among them, autologous plasma can only be added in the early stage of culture, and autologous plasma can no longer be added in the later stage of culture.

[0013] In an alternative embodiment of the first aspect of the present application, among the cytokines, 100-1000 U / mL IL-2, 10-50 ng / mL IL-7, 10-50 ng / mL IL-15, 10-50 ng / mL IL-18, 10-20 ng / mL IL-21 are included.

[0014] The combination of cytokines can stimulate the proliferation of gamma delta T cells, improve the culture environment of gamma delta T cells, improve the culture efficiency and enhance the cell quality, and can enhance the multiple functions of gamma delta T cells, including anti-tumor, anti-infection, immune regulation, etc.

[0015] In an alternative embodiment of the first aspect of the present application, the bisphosphate is one or both of pamidronate disodium and zoledronic acid.

[0016] In the present application, bisphosphate is a cell proliferation additive, pamidronate disodium and / or zoledronic acid, pamidronate disodium and / or zoledronic acid have a significant stimulating expansion effect in PBMC culture of gamma delta T cells. By expanding gamma delta T cells, pamidronate disodium and / or zoledronic acid can help enhance the immune response capacity of the body, thereby playing an important role in the treatment of diseases such as tumors and infections. The concentration of the bisphosphate is 1-10 μg / mL.

[0017] In an alternative embodiment, the bisphosphonate is 5 μg / mL zoledronic acid, and in other embodiments, the bisphosphonate can be 5 μg / mL pamidronate disodium.

[0018] As a second aspect of the present application, the gamma delta T cell culture medium of the present application comprises: a basal medium, cytokines, a bisphosphonate, berberine and autologous plasma, the concentration of the berberine is 0.25-5 μM; the cytokines comprise 100-1000 U / mL IL-2, 10-50 ng / mL IL-7, 10-50 ng / mL IL-15, 10-50 ng / mL IL-18, 10-20 ng / mL IL-21; the bisphosphonate is one of 1-10 μg / mL pamidronate disodium and 1-10 μg / mL zoledronate; the addition amount of the autologous plasma is 5-10%.

[0019] As a third aspect of the present application, the present application further provides a culture method of gamma delta T cells, comprising:

[0020] obtaining cells to be treated;

[0021] culturing the cells to be treated based on the gamma delta T cell culture medium provided by the first aspect of the present application according to a preset culture duration;

[0022] collecting the cells after culture.

[0023] Specifically, in the culture method of gamma delta T cells of the present application, the cells to be treated are PBMCs. The separated PBMCs are inoculated into a culture container and the gamma delta T cell culture medium is added for culture, wherein the inoculation density of the PBMCs is 3-8 x 10 6 per mL.

[0024] Further, during the culture, the medium is added according to the culture condition so as to maintain the cell concentration at 2.0-3.5 x 10 6 per mL.

[0025] Further, the culture duration of the gamma delta T cells is 14 days, autologous plasma is added 9 days before culture, and the concentration of the added autologous plasma is 5-10%, preferably 10%. After 9 days, no additional autologous plasma is added. After the end of culture, the cells are collected and the purity and expansion multiple of the gamma delta T cells are detected.

[0026] The culture container referred to in the present application comprises a culture bottle or a culture bag, such as a T25 or T75 culture bottle, a T175 culture bottle, a T225 culture bottle and a 1L culture bag, etc. According to the actual volume of the culture medium, transfer or replacement can be performed to ensure sufficient space for culture.

[0027] In a fourth aspect of the present application, a kit is provided, the kit comprising the gamma delta T cell culture medium provided by the first aspect of the present application, and the kit is used for culture of gamma delta T cells or / and preparation of exosomes.

[0028] Beneficial effects: the present application provides a kind of γδT cell culture medium, culture method and application, the cell culture medium includes basic culture medium, cytokine, bisphosphonate and berberine, the concentration of the berberine is 0.25 ~ 5 μM.The culture medium with berberine combines other components, the proliferation of γδT cell has promoting effect, not only can improve the survival rate of γδT cell, can also improve the expansion rate of γδT cell more obviously. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the schematic diagram of the influence of different concentrations of berberine on the survival rate of total PBMCs;

[0030] Figure 2 is the schematic diagram of the influence of different concentrations of berberine on the proliferation of various immune cells in PBMCs;

[0031] Figure 3 is the schematic diagram of the expansion fold of γδT cells in examples and comparative examples;

[0032] Figure 4 is the schematic diagram of the flow cytometry detection of the purity of γδT cells in examples and comparative examples. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and effect of the present application more clear and definite, the present application will be further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0034] As the first aspect of the present application, the γδT cell culture medium in the present application is composed of A component, B component, C component and D component:

[0035] The A component is a basic culture medium, including one of AIM-V culture medium, KBM-581 culture medium and RPMI-1640;

[0036] The B component is bisphosphonate, including one of 1 ~ 10 μg / mL pamidronate disodium and 1 ~ 10 μg / mL zoledronate;

[0037] The C component is a cytokine, 100 ~ 1000 U / mL IL-2, 10 ~ 50 ng / mL IL-7, 10 ~ 50 ng / mL IL-15, 10 ~ 50 ng / mL IL-18, 10 ~ 20 ng / mL IL-21;Wherein, the concentration referred to is the number of active units of IL-2 contained in each milliliter of culture medium;

[0038] The D component is berberine, and the concentration of berberine added is 0.25 ~ 5 μM.

[0039] The gamma delta T cell culture medium can further comprise autologous plasma, the autologous plasma is added for 0-9 days before culture, the amount of addition is 5-10%, preferably 10%, and the amount of addition specifically refers to the amount of addition of autologous plasma in the cell culture medium is 5% to 10% of the total volume of the culture medium.

[0040] The kit of the present application, in combination with cytokines and bisphosphates, can promote the survival rate and expansion rate of gamma delta T cells in the culture medium with the addition of berberine.

[0041] The present application also claims a preparation method of a gamma delta T cell culture medium, and the gamma delta T cells obtained based on the preparation method account for more than 60%, and can be as high as 79%.

[0042] The present application also claims the application of the gamma delta T cell culture medium, which can be used in a kit, and the kit is used for the expansion of gamma delta T cells and the preparation of gamma delta T cell exosomes. The corresponding kit can improve the expansion rate of immune cells in vitro, and the immune cell culture kit has relatively low cost, which is convenient for enterprises to save costs.

[0043] In order to better illustrate the technical effects of the technical scheme of the present application, the present application constructs the following examples and comparative examples for testing.

[0044] Example 1: The gamma delta T cell culture medium of example 1 comprises:

[0045] The basic culture medium is AIM-V culture medium;

[0046] The complete culture medium comprises 5 μg / mL zoledronic acid;

[0047] The complete culture medium comprises cytokines, 700 U / mL IL-2, 20 ng / mL IL-7, 150 ng / mL IL-15, 50 ng / mL IL-18, and 10 ng / mL IL-21;

[0048] The complete culture medium comprises 0.25 μM berberine;

[0049] The complete culture medium comprises 10% autologous plasma.

[0050] The culture steps of the gamma delta T cells are as follows:

[0051] S1: isolating human peripheral blood mononuclear cells (PBMCs);

[0052] The isolation method of step S1 is Ficoll density gradient centrifugation;

[0053] S2: inoculating the PBMCs into a culture vessel, and the inoculation density is 3-8 x 10 6cells / mL, and the above complete medium is added for culture;

[0054] S3: During the culture process, an appropriate amount of complete medium is added for culture, and the mixed medium is supplemented in real time according to the cell density and the color of the culture medium during the culture process. The supplementing liquid complies with the principle of maintaining the cell concentration at 2.0-3.5 x 10 6 cells / mL, and the above complete medium is added for culture;

[0055] S4: After the culture is completed, the cells are collected and detected.

[0056] Example 2: The γδ T cell culture medium of Example 2 comprises:

[0057] The basic medium is AIM-V medium;

[0058] The complete medium comprises 5 μg / mL zoledronic acid;

[0059] The complete medium comprises cytokines, 700 U / mL IL-2, 20 ng / mL IL-7, 150 ng / mL IL-15, 50 ng / mL IL-18, and 10 ng / mL IL-21;

[0060] The complete medium comprises 2.5 μM berberine;

[0061] The complete medium comprises 10% autologous plasma.

[0062] The culture method of Example 2 is the same as that of Example 1.

[0063] Example 3: The γδ T cell culture medium of Example 3 comprises:

[0064] The basic medium is AIM-V medium;

[0065] The complete medium comprises 5 μg / mL zoledronic acid;

[0066] The complete medium comprises cytokines, 700 U / mL IL-2, 20 ng / mL IL-7, 150 ng / mL IL-15, 50 ng / mL IL-18, and 10 ng / mL IL-21;

[0067] The complete medium comprises 5 μM berberine;

[0068] The complete medium comprises 10% autologous plasma.

[0069] The culture method of Example 3 is the same as that of Example 1.

[0070] The culture medium of Comparative Example 1 includes:

[0071] The base medium is AIM-V medium;

[0072] The complete medium includes cytokines, 700 U / mL IL-2, 20 ng / mL IL-7, 150 ng / mL IL-15, 50 ng / mL IL-18, 10 ng / mL IL-21;

[0073] The complete medium includes 10% autologous plasma.

[0074] Wherein, the culture method of Comparative Example 1 is the same as that of Example 1.

[0075] The culture medium of Comparative Example 2 includes:

[0076] The base medium is AIM-V medium;

[0077] The complete medium includes 5 μg / mL zoledronic acid;

[0078] The complete medium includes cytokines, 700 U / mL IL-2, 20 ng / mL IL-7, 150 ng / mL IL-15, 50 ng / mL IL-18, 10 ng / mL IL-21;

[0079] The complete medium includes 10% autologous plasma.

[0080] Wherein, the culture method of Comparative Example 2 is the same as that of Example 1.

[0081] Control test experiment

[0082] Test 1: Test the effect of different concentrations of berberine on the survival rate of total PBMCs by using the control variable method

[0083] Test method: Set the concentration of berberine to be 0, 0.2, 0.4, 0.8, 1, 2.5, 5, and 10 μM, respectively, and the complete medium is AIM-V medium containing 10% FBS. PBMCs are cultured using the medium of each group until the 3rd day, and then the CCK8 kit is used to detect the total cell survival rate, and the test results are shown in FIG. 1.

[0084] Figure 1 is a schematic diagram of the survival rate of PBMCs at different concentrations of berberine. As can be seen from the figure, berberine can improve the survival rate of PBMCs, and the increase in the survival rate increases with the increase in the concentration of berberine. When the concentration of berberine is 10 μM, the effect of increasing the survival rate of γδ T cells is not obvious, and the cost is relatively high. Therefore, the concentration of berberine is preferably 0.25-5 μM, and more preferably 2.5-5 μM.

[0085] Test 2: Effect of different concentrations of berberine on the proliferation of various immune cells in PBMCs

[0086] Test method: The concentration of berberine was set to be 0, 0.25, and 5 μM, and the complete culture medium was AIM-V culture medium containing 10% FBS. PBMCs were cultured in each group of culture medium to the 14th day, and then the proportion of each type of immune cell in the total cells was detected by flow cytometry. The results are shown in Figure 2.

[0087] As can be seen from Figure 2, berberine has a relatively obvious effect on promoting the proliferation of double-negative T cells and γδ T cells. Among them, for γδ T cells, when there is no berberine, the proportion of γδ T cells is only 37.2%, while when the concentration of berberine is 0.25 μM, the proportion of γδ T cells reaches 41.3%, and when the concentration of berberine is 5 μM, the proportion of γδ T cells can reach 50.7%.

[0088] Test 3: Cell survival rate detection

[0089] The cells collected at different culture times in Examples 1-3 and Comparative Examples 1-2 were subjected to AO / PI staining, and cell counting was performed using a cell counter to calculate the cell survival rate and the number of cells, and a cell expansion fold graph was drawn, and the results are shown in Figure 3.

[0090] As can be seen from Figure 3, the examples of the present application can improve the cell expansion fold of γδ T cells, and have an expansion effect on the culture of γδ T cells.

[0091] Test 4: γδ T cell purity detection

[0092] The collected total cells were subjected to γδ T cell purity detection. The collected cells were subjected to γδ T purity detection by flow cytometry detection method, and the antibodies used were: PerCP-Cy5.5-CD3 antibody, FITC anti-human TCR γ / δ antibody. γδ T cells are CD3+TCR γ / δ+cell groups. The results are shown in Table 1 and Figure 4.

[0093] Table 1, proportion of γδ T cells in each group (%)

[0094] Group Day 0 Day 14 Comparative Example 1 14.4 ± 0.06 37.63 ± 0.93 Comparative Example 2 14.4 ± 0.06 52.47 ± 0.23 Experimental Example 1 14.4 ± 0.06 62.30 ± 0.61 Experimental Example 2 14.4 ± 0.06 73.80 ± 0.17 Experimental Example 3 14.4 ± 0.06 79.37 ± 0.12

[0095] As can be seen from Table 1 and Figure 4, the culture medium of the embodiment of the present application has a promoting effect on the proliferation of γδ T cells. Within the scope defined by the present application, the addition of berberine can make the proportion of γδ T cells reach more than 60%, and when the addition concentration of berberine is 2.5 μM-5 μM, the proportion of γδ T cells can reach more than 70% after 14 days of culture, and when the addition concentration is 5 μM, the proportion of γδ T cells is close to 80% after 14 days of culture. Therefore, the γδ T cell culture medium of the present application has a promoting effect on the proliferation of γδ T cells, and provides strong support for the subsequent clinical use of γδ T cells.

[0096] The present application also provides a kit comprising the γδ T cell culture medium as described above, which can be applied to the culture of γδ T cells, the preparation of supernatant exosomes and the like.

[0097] The above-described embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements to some technical features thereof; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A gamma delta T cell culture medium, characterized in that, The culture medium comprises: a basic medium, cytokines, a bisphosphonate and berberine, wherein the concentration of the berberine is 0.25-5 μM.

2. The γδ T cell medium of claim 1, wherein, The culture medium of the γδT cells further comprises 5-10% autologous plasma.

3. The γδ T cell medium of claim 1, wherein, The cytokines comprise 100-1000 U / mL IL-2, 10-50 ng / mL IL-7, 10-50 ng / mL IL-15, 10-50 ng / mL IL-18 and 10-20 ng / mL IL-21.

4. The γδ T cell medium of claim 1, wherein, The bisphosphonate is one or both of pamidronate disodium and zoledronic acid, and the concentration of the bisphosphonate is 1-10 μg / mL.

5. The γδ T cell medium of claim 4, wherein, The bisphosphonate is 5 μg / mL zoledronic acid.

6. The γδ T cell medium of claim 1, wherein, The basic medium comprises one of AIM-V medium, KBM-581 medium and RPMI-1640.

7. A gamma delta T cell culture medium, characterized in that, The culture medium comprises: a basic medium, cytokines, a bisphosphonate, berberine and autologous plasma, wherein the concentration of the berberine is 0.25-5 μM; the cytokines comprise 100-1000 U / mL IL-2, 10-50 ng / mL IL-7, 10-50 ng / mL IL-15, 10-50 ng / mL IL-18 and 10-20 ng / mL IL-21; the bisphosphonate is one of 1-10 μg / mL pamidronate disodium and 1-10 μg / mL zoledronic acid; and the autologous plasma is added in an amount of 5-10%.

8. A method for culturing γδ T cells, characterized by, The culture medium comprises: obtaining cells to be treated; culturing the cells to be treated based on the culture medium of the γδT cells according to any one of claims 1-6 for a preset culture duration; and collecting the cultured cells.

9. The culture method of γδ T cells according to claim 8, characterized by, The cells to be treated are PBMCs, and the culture duration is 14 days.

10. A kit characterized in that, The kit comprises the culture medium according to any one of claims 1-6, and is used for culturing γδT cells or / and preparing exosomes.

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