Culture medium, culture method and application thereof for inducing C2 type iNKT cells

Abstract

The present invention provides a culture medium, a culture method and an application thereof for inducing C2 type iNKT cells. The culture medium for inducing C2 type iNKT cells includes IL-2, IL-12, IL-15, IL-18, 7DW8-5, GM-CSF, IL-4, TNF-α, LPS, wogonin, hyperoside, autologous plasma and AIM-V basal culture medium within a limited range. The proportion of C2 type iNKT cells in CD3+T cells obtained by culturing with the culture medium can reach 32.0%, which can achieve efficient induction and effective amplification, provide guarantee for the research and clinical use of C2 type iNKT cells, and the corresponding C2 type iNKT cells can secrete a higher level of IFN-γ without target cell stimulation.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a culture medium for inducing C2 type iNKT cells, a culture method and application thereof. Background Art

[0002] iNKT cells (invariant natural killer T cells) are a type of innate T lymphocytes that are considered to be the first cells to respond to infection. iNKT cells have always been considered to be tissue-resident immune cells that regulate local and systemic immune effects. They secrete a variety of cytokines (such as IFN-γ, IL-4, IL-17, etc.) and chemokines after recognizing lipid antigens presented by CD1d, thereby mediating innate and acquired immune responses. Recent studies have found that iNKT cells have strong tumor cell-killing activity, and activated iNKT cells can reactivate exhausted CD8+T cells and NK cells through the secretion of cytokines.

[0003] According to the expression of CD244 and CXCR6, some scholars have divided iNKT into three subtypes: C0 iNKT, C1 iNKT, and C2iNKT. Among them, C2 shows more NK cell characteristics; C2 iNKT secretes higher levels of IFN-γ, granzyme B, perforin, etc. than C1 iNKT. Studies have shown that C2 iNKT cells circulate in peripheral tissues, inhibit tumor spread, enhance antiviral immune response, and show high cytotoxicity in the human body.

[0004] Some studies now believe that IL-15 can stimulate and promote C2 iNKT cells, but they differentiate into C1 or C2 iNKT cells with the same probability, and the induction efficiency is not very high. Therefore, more efficient induction and expansion of C2 iNKT cell culture media and methods are needed to meet research needs. Summary of the invention

[0005] The present invention provides a culture medium, a culture method and application thereof for inducing C2 type iNKT cells, so as to solve the problem of low induction efficiency of the existing method and realize the efficient induction of C2 type iNKT cells.

[0006] As a first aspect of the present invention, the present invention provides a culture medium for inducing C2 type iNKT cells, the culture medium comprising: 10-1000U / mL IL-2, 1-100ng / mL IL-12, 1-100ng / mL IL-15, 1-100ng / mL IL-18, 10-1000ng / mL7DW8-5, 10-1000ng / mL GM-CSF, 1-100ng / mL IL-4, 10-1000U / mL TNF-α, 1-100ng / mL LPS, 0.1-10μM wogonin, 0.1-10μM hyperoside, 1-10% autologous plasma and AIM-V basal culture medium.

[0007] Preferably, the culture medium comprises: 700U / mL IL-2, 10ng / mL IL-12, 20ng / mL IL-15, 10ng / mL IL-18, 20ng / mL 7DW8-5, 50ng / mL GM-CSF, 10ng / mL IL-4, 100U / mL TNF-α, 20ng / mLLPS, 1μM wogonin, 1μM hyperoside, and 10% autologous plasma.

[0008] As a second aspect of the present invention, the present invention also provides a culture method for inducing C2 type iNKT cells, the method comprising:

[0009] Obtaining cells to be processed;

[0010] Cultivating the cells to be treated based on any of the culture media described above according to a preset culture time;

[0011] The cells to be treated are collected after culture.

[0012] The method for inducing C2 iNKT cells of the present invention further comprises: the inoculation concentration of the cells to be treated is 3.5×10 6 Pieces / mL.

[0013] A further embodiment of the culture method for inducing C2 type iNKT cells of the present invention is as follows: the cells to be processed are PBMCs, and the culture time is 14 days.

[0014] As a third aspect of the present invention, the present invention also claims protection for a kit, which comprises any one of the above-mentioned culture media, and is used to induce the culture of C2 type iNKT cells and / or the preparation of exosomes.

[0015] The present invention provides a culture medium for inducing C2 type iNKT cells, the culture medium comprising IL-2, IL-12, IL-15, IL-18, 7DW8-5, GM-CSF, IL-4, TNF-α, LPS, wogonin, hyperoside, autologous plasma and AIM-V basal culture medium within a limited range. The proportion of C2 type iNKT cells in CD3+T cells obtained by culturing with the culture medium can reach 32.0%, which is about 133 times higher than that before culturing, achieving efficient induction and effective amplification, providing guarantee for the research and clinical use of C2 type iNKT cells; at the same time, the corresponding C2 type iNKT cells can secrete a higher level of IFN-γ without target cell stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The effect of different concentrations of 7DW8-5 on total cell survival;

[0018] Figure 2 The cell morphology during the culture process of Example 1 of the present invention;

[0019] Figure 3 This is the flow cytometry analysis of the proportion of C2 iNKT cells in PBMC of Comparative Example 1;

[0020] Figure 4 This is a flow cytometry graph of the proportion of C2 iNKT cells in Example 1;

[0021] Figure 5 This is a flow cytometry graph of the proportion of C2 iNKT cells in Comparative Example 2;

[0022] Figure 6 This is a flow cytometric analysis of the IFN-γ concentration in the supernatant of PBMCs in Example 1 after 14 days of culture. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0024] After research, the present invention provides a culture medium for inducing C2 type iNKT cells, including: 10-1000U / mL IL-2, 1-100ng / mL IL-12, 1-100ng / mL IL-15, 1-100ng / mL IL-18, 10-1000ng / mL7DW8-5, 10-1000ng / mL GM-CSF, 1-100ng / mL IL-4, 10-1000U / mL TNF-α, 1-100ng / mL LPS, 0.1-10μM wogonin, 0.1-10μM hyperoside, 1-10% autologous plasma and AIM-V basal culture medium.

[0025] Among the components, IL-2 and IL-15 can promote the proliferation and activation of iNKT cells, IL-12 and IL-18 can induce the secretion level of IFN-γ; C0 type iNKT cells and C1 type iNKT cells have the potential to develop into C2 in the presence of IL-15; GM-CSF and IL-4 can induce the proliferation of DC cells; TNF-α and LPS can induce the maturation of DC cells; α-GalCer and 7DW8-5 are lipid antigens that can bind to the CD1d molecules expressed on the surface of DC cells, and α-GalCer and 7DW8-5 can activate iNKT cells, that is, α-GalCer and 7DW8-5 are loaded on the CD1d molecules of DC cells, and then presented by DC cells to iNKT cells, inducing the proliferation and activation of iNKT cells. Autologous plasma in iNKT culture medium also plays a role in providing nutrients. Hyperoside provides nutrients for the culture medium, and hyperoside, wogonin and 7DW8-5 can jointly promote the proliferation of iNKT cells and the secretion of IFN-γ.

[0026] Among them, the AIM-V basic culture medium can also be replaced according to actual selection needs.

[0027] In order to better illustrate the technical effect of the technical solution of the present invention, the present invention constructs the following embodiments and comparative examples for experiments.

[0028] Example 1

[0029] The culture medium for inducing C2 type iNKT cells in this Example 1 includes the following components: 700U / mL IL-2, 10ng / mL IL-12, 20ng / mL IL-15, 10ng / mL IL-18, 20ng / mL 7DW8-5, 50ng / mL GM-CSF, 10ng / mL IL-4, 100U / mL TNF-α, 20ng / mL LPS, 1μM wogonin, 1μM hyperoside, 10% autologous plasma, and AIM-V basal medium.

[0030] Wherein, its culture method comprises the steps:

[0031] S10: obtaining cells to be processed;

[0032] Specifically, peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral venous blood using density gradient centrifugation.

[0033] S20: culturing the PBMC cells obtained in step 1 using the culture medium of Example 1 according to a preset culture time;

[0034] Specifically, the specific operation of step S20 is: resuspend the PBMC cells in complete culture medium preheated at 37°C and inoculate them in a 6-well plate at a cell concentration of 3.5×10 6 / mL, 2mL / well. Then culture in a 37℃ carbon dioxide incubator, and perform fluid replenishment / replacement every 2-3 days according to the cell proliferation, for a total of 14 days.

[0035] S30: collecting the cultured cells to be treated;

[0036] Specifically, after the culture is completed, all cells are collected.

[0037] Comparative Example 1

[0038] Comparative Example 1 is peripheral blood mononuclear cells (PBMC), which are not cultured.

[0039] Comparative Example 2

[0040] Compared with Example 1, the culture medium composition of Comparative Example 2 only contains 700U / mL IL-2, 10ng / mL IL-12, 20ng / mL IL-15, 10ng / mL IL-18, 50ng / mL GM-CSF, 10ng / mL IL-4, 100U / mL TNF-α, 20ng / mL LPS, 10% autologous plasma and AIM-V basal medium, but does not contain 7DW8-5, baicalin and hyperoside.

[0041] Among them, the cultivation method of comparative example 2 is the same as that of embodiment 1.

[0042] Experiment 1

[0043] Different 7DW8-5 concentrations were set, and a single variable was controlled to verify the effects of different concentrations of 7DW8-5 treatment on the survival of total cells (including iNKT cells) obtained after 14 days of culture (culture steps and other conditions were the same as in Example 1). The results are shown in the figure Figure 1 shown.

[0044] from Figure 1It can be seen that 7DW8-5, under the premise of ensuring that other components remain unchanged, different concentrations of 7DW8-5 have different effects on the total cell survival rate. 7DW8-5 can significantly improve the cell survival rate at 10-1000ng / mL, among which 20ng / mL can significantly improve the cell survival rate without increasing the dosage cost too much.

[0045] Experiment 2

[0046] Peripheral blood mononuclear cells (PBMC) were extracted from peripheral blood using the Ficoll method (density gradient centrifugation), and the survival rate of PBMC was detected. Then iNKT cells were cultured, and the morphology and purity of iNKT cells and the survival rate of total cells were detected after 14 days of culture.

[0047] The cell morphology during PBMC culture in Example 1 is shown in FIG. Figure 2 As shown, from Figure 2 It can be concluded that from the 5th day, the cells began to aggregate and show a significant expansion effect.

[0048] The method for detecting PBMC and total cell viability after 14 days of culture is as follows:

[0049] The collected PBMC or total cells were stained with AO / PI, and the cells were counted using a cell counter to calculate the cell survival rate, as shown in Table 1.

[0050] Table 1. PBMC and total cell survival rate after 14 days of culture (%)

[0051] Group PBMC Total cells obtained after 14 days of culture Example 1 98.24±0.39 90.99±1.46 Comparative Example 1 98.24±0.39 No training Comparative Example 2 98.24±0.39 83.75±1.56

[0052] It can be seen from Table 1 that, compared with Comparative Example 2, the culture medium of Example 1 used in the present invention can still achieve a total cell survival rate of more than 90% after 14 days of culture, and a relatively high number of surviving cells are still present.

[0053] C2 iNKT cells (CD3 + CXCR6 + CD244 + )The purity detection method is as follows:

[0054] After 14 days of PBMC culture, total cells were collected for C2 iNKT cells (CD3 + CXCR6 + CD244 + ) purity detection. Flow cytometry was used to detect the C2 iNKT cells (CD3 + CXCR6 + CD244 +) was tested for purity, and the antibodies used were: PerCP-Cy5.5-CD3 antibody, FITC-CD244 antibody, APC-CXCR6 antibody, and the expression of C2 iNKT cells was CD3 + CXCR6 + CD244 + The C2 iNKT cells in the examples and comparative examples accounted for CD3 + The flow cytometry analysis of the proportion of T cells is shown in Figure 3-5 shown.

[0055] Figure 3 This is a flow cytometry chart of the proportion of C2 iNKT cells in Example 1 of the present invention. Figure 3-5 It can be seen that the proportion of C2 type iNKT cells in CD3+T cells obtained after 14 days of culture in the culture medium of the present invention can be 32.0%, which is about 133 times larger than the 0.24% of C2 type iNKT cells in PBMCs, and close to 5 times the 6.56% in Comparative Example 2. The above results show that the culture medium of Example 1 of the present invention can achieve effective expansion of C2 type iNKT cells, providing a guarantee for subsequent research and clinical use of C2 type iNKT cells.

[0056] Experiment 3

[0057] Flow cytometry was used to detect cytokines in the collected supernatant using two fluorescent pairs of PE and APC, and the component content of Example 11 was tested, specifically the content of cytokines IFN-γ, TNF-α, IL-8, IL-4, IL-12p70 and IL-17 in the cell supernatant. The specific test results are shown in Figure 6 shown.

[0058] Figure 6 In, from Figure 6 It can be seen that the IFN-γ level of Example 1 of the present invention is as high as 565.92 pg / mL. From the results, it can be concluded that the C2 type iNKT cells obtained after 14 days of culture in the medium of the present invention can secrete a high level of IFN-γ without target cell stimulation, which is much higher than the existing patents such as patent CN116004533 A (secreted IFN-γ level <150 pg / mL).

[0059] In summary, the culture medium for inducing C2 iNKT cells of the present invention can efficiently induce the proliferation of C2 iNKT cells and expand the number of C2 iNKT cells, and the cultured C2 iNKT cells are rich in IFN-γ factor.

[0060] The present invention also provides a kit, which includes the culture medium for inducing C2 type iNKT cells as described above, and the kit can be applied to C2 type iNKT cell culture, supernatant exosome preparation and other aspects.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for inducing C2 type iNKT cells, characterized in that: The culture medium is composed of the following components: 10-1000 U / mL IL-2, 1-100 ng / mL IL-12, 1-100 ng / mL IL-15, 1-100 ng / mL IL-18, 10-1000 ng / mL 7DW8-5, 10-1000 ng / mL GM-CSF, 1-100 ng / mL IL-4, 10-1000 U / mL TNF-α, 1-100 ng / mL LPS, 0.1-10 μM wogonin, 0.1-10 μM hyperoside, 1-10% autologous plasma and AIM-V basal culture medium.

2. The culture medium for inducing C2 type iNKT cells according to claim 1, characterized in that The culture medium consists of the following components: 700 U / mL IL-2, 10 ng / mL IL-12, 20 ng / mL IL-15, 10 ng / mL IL-18, 20 ng / mL 7DW8-5, 50 ng / mL GM-CSF, 10 ng / mL IL-4, 100 U / mL TNF-α, 20 ng / mL LPS, 1 μM wogonin, 1 μM hyperoside, 10% autologous plasma and AIM-V basal medium.

3. A method for inducing C2 type iNKT cells, characterized in that: The method comprises: Obtaining cells to be processed; Cultivating the cells to be treated based on the culture medium according to any one of claims 1 to 2 according to a preset culture time; collecting the cells to be treated after culture; The cells to be treated are PBMCs, and the culture period is 14 days.

4. The method for inducing C2 type iNKT cells according to claim 3, characterized in that: The cell concentration to be treated is 3.5×10 6 Pieces / mL.

5. A kit, characterized in that: The kit comprises the culture medium according to any one of claims 1 to 2, and is used to induce the culture of C2 type iNKT cells and / or the preparation of exosomes.

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