A human Vγ9Vδ2T cell expansion culture medium and expansion culture method thereof

Abstract

The present invention discloses a kind of human Vγ9Vδ2T cell expansion culture medium and its culture method, belong to the field of cell culture technology, culture medium includes Optimizer serum-free culture medium, also includes interleukin-2 and interleukin-15 and N-acetylcysteine. Based on the culture medium amplification of human Vγ9Vδ2T cells, it is possible to significantly reduce the ROS produced in cell amplification, improve the proliferation efficiency and cell purity of human Vγ9Vδ2T cells, and the human Vγ9Vδ2T cells obtained by culture have stronger killing and inhibitory abilities to tumors, and the cell anti-apoptosis ability is stronger. And the culture method is suitable for large-scale amplification, so as to provide a solid foundation for the clinical use of human Vγ9Vδ2T cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a human Vγ9Vδ2T cell expansion culture medium and an expansion culture method thereof. Background Art

[0002] Adoptive tumor immunotherapy involves transferring sensitized lymphocytes (with specific immunity) or their products to individuals with compromised cellular immunity (such as cancer patients) to enhance anti-tumor immunity. In 1985, the U.S. National Cancer Research Council established cancer immunotherapy as the fourth treatment option, following surgery, radiotherapy, and chemotherapy.

[0003] T cells can be divided into two categories based on the type of TCR on their surface: αβT cells and γδT cells. αβT cells, commonly known as T cells, express the TCRαβ on their surface and account for over 95% of all T cells. γδT cells, on the other hand, express the TCRγδ on their surface. γδT cells, discovered in 1986, possess the ability to rapidly and directly respond in the initial stages of disease and play a crucial role in innate immune responses. γδT cells, a major T cell subset distinct from αβT cells, serve as a bridge between innate and adaptive immunity in the human body, participating in a variety of immune responses and playing a crucial role in tumor immunity, infection immunity, and autoimmune regulation. γδT cells exert direct cytotoxicity against tumor cells, controlling their growth and metastasis through both direct killing and cytokine release. Their killing activity is independent of MHC molecules. A Nature Medicine study reported that a large-scale analysis of 9,020 patients with various cancers demonstrated that intratumoral γδT cell infiltration is the best prognostic marker for all tumors. Therefore, more and more researchers are paying attention to the anti-tumor effect of γδT.

[0004] Human γδT cells can be divided into Vδ1, Vδ2, and Vδ3 T cells based on their surface δ chains. Typically, 50% to 75% of γδT lymphocytes in peripheral blood express the Vδ2 chain, along with the Vγ9 chain. These cells are designated human Vγ9Vδ2 T cells. Because human Vγ9Vδ2 T cells predominate among peripheral blood γδT cells, recognize phosphorylated antigens, and possess potent anti-tumor activity, most clinical research on γδT cells has focused on human Vγ9Vδ2 T cells. Due to their low proportion in peripheral blood, existing expansion methods suffer from low in vitro expansion multiples, resulting in insufficient numbers of cells for clinical use. Commonly used expansion methods involve activation with TCRγδ antibodies or aminophosphodiesterases, supplemented with cytokines such as IL-2. However, γδT cells amplified using TCRγδ antibodies contain both Vδ1 and Vδ2 subsets. Furthermore, antibody-based amplification is costly, results in low numbers of cells amplified, and therefore cannot be scaled up to meet clinical needs. Another existing amplification route, using aminophosphates such as zoledronic acid for amplification, will lead to the accumulation of ROS, resulting in a shorter survival time of the amplified cells, weak anti-apoptosis ability, weak cytokine secretion ability, and weak tumor-killing ability. Summary of the Invention

[0005] The main purpose of the present invention is to overcome the shortcomings of the above-mentioned existing technologies and provide a method for expanding and culturing human Vγ9Vδ2T cells with significantly enhanced anti-apoptosis ability and tumor killing ability, and its culture medium. In addition, the culture method is a serum-free large-scale culture scheme that can meet subsequent clinical needs.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] A human Vγ9Vδ2 T cell expansion medium comprises an OPTMizer serum-free medium, interleukin-2, interleukin-15 and N-acetylcysteine.

[0008] Preferably, the concentration of interleukin-2 is 100-1000 U / ml, the concentration of interleukin-15 is 1-100 ng / ml, and the concentration of N-acetylcysteine ​​is 100 μM-10 mM.

[0009] Preferably, aminophosphoric acid compounds are added to the above-mentioned human Vγ9Vδ2T cell expansion medium to obtain the corresponding human Vγ9Vδ2T cell activation medium.

[0010] Preferably, in the above-mentioned human Vγ9Vδ2T cell activation culture medium, the aminophosphoric acid compound includes one or more of zoledronic acid, pamidronic acid, bromopropane diphosphate, and isopentenyl pyrophosphate; and the concentration of the aminophosphoric acid compound is 1-100 μM.

[0011] A culture method based on the above-mentioned human Vγ9Vδ2 T cell expansion medium comprises the following steps:

[0012] Step 1: activating and culturing peripheral blood mononuclear cells to obtain an activated cell suspension;

[0013] Step 2: Dilute the activated cell suspension with human Vγ9Vδ2 T cell expansion medium and continue culturing for 72 hours;

[0014] Step 3: Add human Vγ9Vδ2 T cell expansion medium and continue culturing for 72 hours;

[0015] Step 4: Transfer the obtained culture medium to a culture container and add human Vγ9Vδ2 T cell expansion medium;

[0016] Step 5: Continue culturing for 72 hours;

[0017] Step 6: Collect cells.

[0018] Preferably, the activation culture in step 1 comprises resuspending the peripheral blood mononuclear cells in human Vγ9Vδ2 T cell activation medium and culturing them statically for 72 hours.

[0019] Preferably, the cell suspension obtained in step 1 has a cell concentration of 1-2*10 6 pieces / mL.

[0020] Preferably, the dilution in step 2 is to a cell concentration of 0.5-1*10 6 pieces / mL.

[0021] Preferably, the culture in steps 2-3 is carried out in a T175 culture flask. During steps 2-3, the cell culture fluid is added with human Vγ9Vδ2 T cell expansion medium every 48-72 hours, and the amount of added fluid is sufficient to replenish the cell culture fluid to 2 / 3 of the flask volume.

[0022] Preferably, transferring the culture container in step 4 includes transferring the culture medium from the T175 culture flask into a 1.8 L cell culture bag, and in step 4, human Vγ9Vδ2 T cell expansion medium is added to 1000 mL.

[0023] Preferably, the above step 3 comprises: adding human Vγ9Vδ2 T cell expansion medium and paclitaxel, and continuing to culture for 72 hours;

[0024] Preferably, the above step 4 comprises: transferring the obtained culture medium to a culture container, adding human Vγ9Vδ2 T cell expansion medium, and adding TGF-β.

[0025] Preferably, in step 3, the final concentrations of the components added are N-acetylcysteine ​​to a final concentration of 15-19 mM and paclitaxel to a final concentration of 0.5-1 μM.

[0026] Preferably, the final concentrations of the components added in step 4 are N-acetylcysteine ​​to a final concentration of 18-20 mM and TGF-β to a final concentration of 4-7 nM.

[0027] Beneficial effects of the present invention:

[0028] The purpose of the present invention is first to provide a method for expanding and culturing human Vγ9Vδ2T cells, which can improve the proliferation efficiency and cell purity of human Vγ9Vδ2T cells. The cultured human Vγ9Vδ2T cells have strong tumor killing and inhibitory abilities, and the cells themselves survive longer and have stronger anti-apoptosis abilities. This method is based on the development of a serum-free expansion medium for human Vγ9Vδ2T cells, which can selectively expand human Vγ9Vδ2T cells from peripheral blood mononuclear cells, and compared with the traditional RPMI+10% fetal bovine serum culture method, the expanded human Vγ9Vδ2T cells have high purity. In addition, the expansion method provided by the present invention is a large-scale expansion method with a culture volume of up to 2L, which greatly increases the number of expanded human Vγ9Vδ2T cells, thereby providing a solid foundation for the clinical use of human Vγ9Vδ2T cells.

[0029] Specifically, the addition of N-acetylcysteine ​​to the human Vγ9Vδ2T cell proliferation culture medium effectively prevents cell apoptosis; the addition of paclitaxel and TGF-β in the preparation method can significantly increase the final expansion multiple and improve the cell purity to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1-2 After 14 days of PBMC expansion, the purity of human Vγ9Vδ2 T cells was detected by flow cytometry. Figure 1 For Example 1, Figure 2 For comparative examples 2-7;

[0031] Figure 3 shows the expansion folds of human Vγ9Vδ2 T cells using different cytokines, with A showing Example 1 and Comparative Example 1, and B showing Examples 1-2 and Comparative Examples 2-7;

[0032] Figure 4 Comparison of ROS in human Vγ9Vδ2 T cells obtained after 14 days of expansion with different cytokines;

[0033] Figure 5 Comparison of apoptosis rates of human Vγ9Vδ2T cells obtained on day 14 after expansion and culture with different cytokines;

[0034] Figure 6Comparison of the killing effects of human Vγ9Vδ2 T cells obtained after expansion and culture with different cytokines on four tumor cell lines: MB231, SW480, huh7, and A375;

[0035] In order to more intuitively reflect the results, in the drawings of the present specification, "IL-2+IL-15" is used to represent Comparative Example 1, and "IL-2+IL-15+NAC" is used to represent Example 1, wherein "NAC" represents N-acetylcysteine, hereinafter referred to as "NAC"; "IL" represents interleukin. DETAILED DESCRIPTION

[0036] Prepare for the experiment:

[0037] The method for extracting peripheral blood mononuclear cells comprises the following steps:

[0038] (1) Transfer peripheral blood to a 50 mL centrifuge tube and centrifuge at 2500 rpm for 10 min;

[0039] (2) Discard the upper plasma layer, dilute the blood cells with normal saline to a ratio of 1:1, and mix thoroughly;

[0040] (3) Pipette 15 mL of Ficoll lymphocyte separation solution into a 50 mL centrifuge tube and slowly and evenly add the mixed cell suspension to the upper layer to form a complete interface;

[0041] (4) Centrifuge at 800 g for 20 min until distinct stratification is observed;

[0042] (5) Collect mononuclear cells and wash twice with PBS;

[0043] (6) Resuspend the cells in PBS and count them to obtain a peripheral blood mononuclear cell suspension for later use.

[0044] The human Vγ9Vδ2T cell activation medium used in the embodiments of the present invention and the comparative examples includes an opTmizer serum-free medium, zoledronic acid, IL-2, IL-15, and NAC. The concentration of zoledronic acid in the human Vγ9Vδ2T cell activation medium is 100 μM, the concentration of IL-2 is 700 U / mL, the concentration of IL-15 is 90 ng / ml, and the concentration of NAC is 1 mM. The human Vγ9Vδ2T cell expansion medium includes an opTmizer serum-free medium, IL-2, IL-15, and NAC are added. The concentration of IL-2 in the human Vγ9Vδ2T cell expansion medium is 500 U / mL, the concentration of IL-15 is 60 ng / ml, and the concentration of NAC is 1 mM. The opTmizer serum-free medium was purchased from Thermo Fisher Scientific with the catalog number A1048501.

[0045] Example 1 A method for culturing human Vγ9Vδ2 T cells:

[0046] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0047] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-0.6*10 6 / mL, static culture for 3 days;

[0048] (3) adding fresh human Vγ9Vδ2 T cell expansion medium and culturing for 3 days;

[0049] The culture in steps 2-3 is performed in a T175 culture flask. During steps 2-3, the cell culture medium is added with human Vγ9Vδ2 T cell expansion medium every 48 hours to a volume that is 2 / 3 of the flask volume.

[0050] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag and add new human Vγ9Vδ2 T cell expansion medium to 1000 mL;

[0051] (5) culturing the culture solution prepared in step (4) for 3 days;

[0052] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation at 400 g for 10 min, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0053] Example 2 A method for culturing human Vγ9Vδ2 T cells:

[0054] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0055] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-0.6*10 6 / mL, static culture for 3 days;

[0056] (3) Add fresh human Vγ9Vδ2 T cell expansion medium and paclitaxel to a final concentration of 0.7 μM, and culture statically for 3 days;

[0057] The culture in steps 2-3 is performed in a T175 culture flask. During steps 2-3, the cell culture medium is added with human Vγ9Vδ2 T cell expansion medium every 48 hours to a volume that is 2 / 3 of the flask volume.

[0058] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag, add new human Vγ9Vδ2 T cell expansion medium to 1000 mL, and add TGF-β to a final concentration of 6 nM;

[0059] (5) culturing the culture solution prepared in step (4) for 3 days;

[0060] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation under the conditions of Example 1, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0061] The difference between Comparative Example 1 and Example 1 is:

[0062] The NAC component was omitted from all culture media.

[0063] Comparative Example 2: A method for culturing human Vγ9Vδ2 T cells:

[0064] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0065] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-0.6*10 6 / mL, static culture for 3 days;

[0066] (3) Add fresh human Vγ9Vδ2 T cell expansion medium, add paclitaxel to a final concentration of 0.7 μM, and culture for 3 days;

[0067] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag and add new human Vγ9Vδ2 T cell expansion medium to 1000 mL;

[0068] (5) culturing the culture solution prepared in step (4) for 3 days;

[0069] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation under the conditions of Example 1, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0070] Comparative Example 3: A method for culturing human Vγ9Vδ2 T cells:

[0071] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0072] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-0.6*10 6 / mL, paclitaxel was added to a final concentration of 0.7 μM, and cultured statically for 3 days;

[0073] (3) adding fresh human Vγ9Vδ2 T cell expansion medium and culturing for 3 days;

[0074] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag and add new human Vγ9Vδ2 T cell expansion medium to 1000 mL;

[0075] (5) culturing the culture solution prepared in step (4) for 3 days;

[0076] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation according to the conditions of Example 1, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0077] Comparative Example 4: A method for culturing human Vγ9Vδ2 T cells:

[0078] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0079] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-0.6*10 6 / mL, static culture for 3 days;

[0080] (3) adding fresh human Vγ9Vδ2 T cell expansion medium and culturing for 3 days;

[0081] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag, add new human Vγ9Vδ2 T cell expansion medium to 1000 mL, and add TGF-β to a final concentration of 6 nM;

[0082] (5) culturing the culture solution prepared in step (4) for 3 days;

[0083] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation according to the conditions of Example 1, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0084] Comparative Example 5: A method for culturing human Vγ9Vδ2 T cells:

[0085] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0086] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-0.6*10 6 / mL, TGF-β was added to a final concentration of 6 nM, and cultured statically for 3 days;

[0087] (3) adding fresh human Vγ9Vδ2 T cell expansion medium and culturing for 3 days;

[0088] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag and add new human Vγ9Vδ2 T cell expansion medium to 1000 mL;

[0089] (5) culturing the culture solution prepared in step (4) for 3 days;

[0090] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation according to the conditions of Example 1, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0091] Comparative Example 6: A method for culturing human Vγ9Vδ2 T cells:

[0092] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0093] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-1*10 6 / mL, static culture for 3 days;

[0094] (3) Add fresh human Vγ9Vδ2 T cell expansion medium, add TGF-β to a final concentration of 6 nM, and culture statically for 3 days;

[0095] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag and add new human Vγ9Vδ2 T cell expansion medium to 1000 mL;

[0096] (5) culturing the culture solution prepared in step (4) for 3 days;

[0097] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation according to the conditions of Example 1, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0098] Comparative Example 7: A method for culturing human Vγ9Vδ2 T cells:

[0099] (1) Peripheral blood mononuclear cells were isolated from peripheral blood and resuspended in human Vγ9Vδ2 T cell activation medium to a cell concentration of 1*10 6 / mL, static culture for 3 days;

[0100] (2) Add human Vγ9Vδ2 T cell expansion medium and dilute the cell concentration to 0.5-1*10 6 / mL, static culture for 3 days;

[0101] (3) Add fresh human Vγ9Vδ2 T cell expansion medium, add TGF-β to a final concentration of 6 nM, and culture statically for 3 days;

[0102] (4) Transfer the culture medium prepared in step (3) to a 1.8 L cell culture bag, add new human Vγ9Vδ2 T cell expansion medium to 1000 mL, and add paclitaxel to a final concentration of 0.7 μM;

[0103] (5) culturing the culture solution prepared in step (4) for 3 days;

[0104] (6) Count the cells and take 1*10 6 The cells were collected by centrifugation according to the conditions of Example 1, and the percentage of CD3+Vδ2+ cells was detected by flow cytometry.

[0105] Experimental testing

[0106] 1. Cell counting

[0107] (1) The cells to be tested were dispersed into single cells, diluted as needed, and a 10 μl volume was selected for relative counting using a flow cytometer;

[0108] (2) After counting is completed, the number of human Vγ9Vδ2 T cells is obtained by dividing the measured number N by 100*dilution factor*cell purity;

[0109] 2. Flow cytometry detection of Vδ2 cell purity

[0110] Immunophenotyping of human Vγ9Vδ2 T cells, including the following steps:

[0111] (1) Take the cultured cells, transfer them into a flow cytometry tube, add 3 mL of PBS and mix thoroughly. Wash the cells by centrifugation (centrifugation conditions: 1600 rpm, 5 min). Pour off the supernatant and suspend the cells in an appropriate amount of PBS according to the concentration and volume of the cell sample to adjust the cell concentration to 1 × 105 cells / mL.

[0112] (2) Fluorescently labeled cells: All examples, comparative examples, and blank controls were transferred to corresponding test tubes, with approximately 5×105 cells per tube. Fluorescent antibody staining solutions were prepared for the control and test tubes according to the antibody instructions: anti-human CD3-V500 and anti-human TCR-Vδ2-PE. Resuspend the cells with 100 μL of the prepared fluorescent antibody staining solution, place in a 4°C refrigerator or on ice, and stain for 15-20 minutes in the dark. Add the corresponding fluorescent antibody to each tube, with 5 μL of each antibody, and add 100 μL of the cell sample suspension to each tube.

[0113] (3) Gently rotate on a vortex shaker to mix thoroughly and incubate at room temperature in the dark for 15 min;

[0114] (4) Wash with 3 mL of PBS, centrifuge at 1600 rpm for 5 min, discard the supernatant, add 500 μl of PBS to suspend the precipitate, and mix thoroughly;

[0115] (5) Test on the machine, the results are shown in Figure 1 、 Figure 2 , Figure 2 The broken line of comparative example 2 almost coincides with that of comparative example 4, and the broken lines of comparative examples 5, 6, and 7 cross each other.

[0116] 3. Calculation of human Vγ9Vδ2 T cell expansion fold

[0117] Human Vγ9Vδ2T cells were cultured with different cytokines, and the cells in culture were counted on D0, D3, D6, D9, D12, D15, and D18, and the purity of the cells at each time point was determined. The number of human Vγ9Vδ2T cells at each time point was obtained by multiplying the number of cells by the purity of human Vγ9Vδ2T cells. The number of human Vγ9Vδ2T cells was divided by the number of cells at D0 to obtain the cell expansion multiple.

[0118] Human Vγ9Vδ2T cells were expanded using culture medium, and the data obtained showed that the cell expansion multiple of the culture medium without NAC addition (Comparative Example 1IL2+IL-15) was low, while the cell expansion multiple of the culture medium with NAC addition (IL2+IL15+NAC) was high. It can be seen that the addition of NAC can significantly improve the proliferation efficiency of human Vγ9Vδ2T cells; however, some current studies have shown that the role of NAC in antioxidant and anti-inflammatory may be related to immune cells, but its mechanism of action may be complex and can affect the immune response through multiple pathways. It is currently disclosed that it may reduce the immune response by reducing oxidative stress, inhibiting the activity of inflammatory cells, and affecting cytokine production. This may include inhibiting T cell activity to a certain extent, and substances that inhibit T cell activity are generally considered to have no effect on promoting T cell proliferation. The expansion folds of Example 2 and Comparative Example 2 were significantly higher than those of Example 1, while no significant change was observed in the expansion fold of Comparative Example 3 compared to Example 1. The addition of NAC and paclitaxel, when added to the expansion system at a specific time, significantly affected the expansion fold. The expansion fold of Comparative Example 4 was significantly higher than that of Example 1, while the expansion folds of Comparative Examples 5, 6, and 7 were lower than those of Example 1. The addition of TGF-β, when added to the expansion system of the present invention at a specific time, had a slight positive effect on the expansion fold, but had a negative effect on the expansion fold when added at other times. Paclitaxel is not currently widely used in cell culture, but existing literature indicates that TGF-β has a certain degree of inhibitory effect on cell proliferation in certain other culture systems. We attempted to add an equivalent amount of paclitaxel to that of Example 2 in step 3 (Comparative Example 2) or an equivalent amount of TGF-β to that of Example 2 in step 4 (Comparative Example 4), based on Example 1. The results showed that either alone had minimal effect on the final expansion fold. Based on our experimental results, we believe that only when the expansion system includes NAC and the combination of paclitaxel or TGF-β is added during a specific cell proliferation period can the expansion multiple be positively affected. We attribute this to the synergistic effect of these two components with each other or with NAC. We will continue to study the specific mechanism in subsequent work. Figure 1 、 Figure 2 It can also be seen that the effects of various substances in the scheme on cell purity are basically consistent with the amplification multiples.

[0119] 4. Flow cytometry detection of ROS content in human Vγ9Vδ2T cells

[0120] (1) Collect cell samples and adjust the cell concentration of the sample to 5*10 5 cells / mL;

[0121] (2) The experiments were divided into positive control, negative control, and experimental groups. The positive control group used 500 μM tert-butyl peroxide (TBHP) to induce oxidative stress. The negative control group added 500 μM NAC before adding TBHP.

[0122] (3) Use a final concentration of 500-1000nM Add the reagent to the sample and incubate at 37°C in the dark for 30-60 minutes;

[0123] (4) Before the last 15 minutes of staining, add 1 μL of 5 μM per 1 ml of appropriate sample. RED Dead Cell Staining Solution, gently mix. The final labeling concentration for staining is 5 nM;

[0124] (5) Analyze samples immediately by flow cytometry.

[0125] Human Vγ9Vδ2T cells were expanded using culture medium. The data obtained showed that the ROS accumulation level of cells in the culture medium without NAC (IL2+IL-15) was higher than that of cells in the culture medium with NAC (IL2+IL15+NAC). This shows that the addition of NAC can significantly reduce the accumulation of ROS in human Vγ9Vδ2T cells during expansion. Some studies have shown that paclitaxel can increase ROS accumulation to a certain extent, but no significant effect was found within the framework of this application. The effect of TGF-β on ROS accumulation is currently uncertain, and similarly, no significant effect was found within the framework of this application.

[0126] 5. Flow cytometry detection of human Vγ9Vδ2T cell apoptosis

[0127] Apoptosis detection of human Vγ9Vδ2 T cells, including the following steps:

[0128] (1) Wash the cells twice with PBS and then resuspend the cells with Annexin V binding buffer at a concentration of 0.25-1.0*10 7 cells / mL;

[0129] (2) Transfer 100 μL of cell suspension into a 5 mL test tube;

[0130] (3) Add 5 μL each of FITC and Annexin V;

[0131] (4) Add 10 μL of PI solution;

[0132] (5) Gently resuspend the cells and incubate in the dark at room temperature for 15 minutes;

[0133] (6) Add 400 μL of binding buffer to each tube and perform flow cytometry analysis.

[0134] Human Vγ9Vδ2T cells were expanded using culture medium. The data obtained showed that the percentage of apoptotic cells in the culture medium without NAC (IL2+IL-15) was higher than that in the culture medium with NAC (IL2+IL15+NAC). This shows that the addition of NAC can significantly reduce apoptosis in human Vγ9Vδ2T cells. The mechanism of the effect of paclitaxel and TGF-β on cell apoptosis is also very complex, with both positive and negative effects. However, no significant effect was found within the framework of this application scheme.

[0135] 6. LDH detection of human Vγ9Vδ2T cell tumorigenicity

[0136] (1) Pipette 50 μL of tumor cells diluted with culture medium into a 96-well plate to a cell count of 5000 per well and culture in a 37°C CO2 incubator overnight to allow the cells to adhere.

[0137] (2) On the second day, 50 μL of human Vγ9Vδ2 T cells were added to the tumor cells in the experimental wells;

[0138] (3) Culture in a 37°C CO2 incubator for 6 hours;

[0139] (4) After adding 10 μL of lysis buffer to the high control wells, the cells were incubated in a 37°C CO2 incubator for 30 min;

[0140] (5) Add 100 μL of working solution to each well and incubate in the dark at room temperature.

[0141] (6) After adding 50 μL of stop solution to each well, immediately measure the absorbance at 490 nm using a microplate reader.

[0142] Human Vγ9Vδ2 T cells cultured with different culture factors were subjected to an LDH release-based killing experiment against four tumor cell lines. The data showed that cells cultured in a medium without NAC (IL2+IL-15) had a lower killing efficiency than cells cultured in a medium supplemented with NAC (IL2+IL15+NAC). This suggests that the addition of NAC can enhance the killing efficiency of human Vγ9Vδ2 T cells. Furthermore, we found that the addition of paclitaxel did not improve the killing efficiency, and TGF-β also had no significant effect on this parameter.

[0143] The preferred specific implementation modes and embodiments of the present invention are described in detail above, but the present invention is not limited to the above implementation modes and embodiments. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the concept of the present invention.

Claims

1. A method for expanding and culturing human Vγ9Vδ2 T cells based on a human Vγ9Vδ2 T cell expansion medium, characterized in that: The following steps are involved: Step 1: activating and culturing peripheral blood mononuclear cells to obtain an activated cell suspension; Step 2: Dilute the activated cell suspension with human Vγ9Vδ2 T cell expansion medium and continue culturing for 72 hours; Step 3: Add human Vγ9Vδ2 T cell expansion medium and paclitaxel to a final concentration of 0.5-1µM and continue culturing for 72 hours; Step 4: Transfer the obtained culture medium to a culture container, add human Vγ9Vδ2 T cell expansion medium, and add TGF-β to a final concentration of 4-7 nM; Step 5: Continue culturing for 72 hours; Step 6: Collect cells; The human Vγ9Vδ2 T cell expansion medium includes an OpTmizer serum-free medium, interleukin-2, interleukin-15, and N-acetylcysteine; the concentration of interleukin-2 is 100-1000 U / ml, the concentration of interleukin-15 is 1-100 ng / ml, and the concentration of N-acetylcysteine ​​is 100 µM-10 mM; An aminophosphoric acid compound is added to the human Vγ9Vδ2T cell expansion medium to obtain a corresponding human Vγ9Vδ2T cell activation medium; in the human Vγ9Vδ2T cell activation medium, the aminophosphoric acid compound includes one or more of zoledronic acid, pamidronate, bromopropane diphosphate, and isopentenyl pyrophosphate; the concentration of the aminophosphoric acid compound is 1-100µM; the activation culture in step 1 includes resuspending peripheral blood mononuclear cells in the human Vγ9Vδ2T cell activation medium and culturing them statically for 72 hours.

2. The method for expanding and culturing human Vγ9Vδ2 T cells according to claim 1, wherein: The transfer of the culture container in step 4 includes transferring the obtained culture solution from the T175 culture flask into a 1.8 L cell culture bag, and adding human Vγ9Vδ2 T cell expansion medium to 1000 mL in step 4.

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