A staged precise regulation in vitro expansion culture method of high-activity γδ T cells

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a staged precise regulation in vitro expansion culture method of high-activity gamma delta T cells. In the synergistic activation stage, the complex activator containing zoledronic acid, IL-2, IL-15, IL-12 and IL-21 is used to significantly improve the early activation level of the gamma delta T cells; in the dynamic expansion stage, the concentration of metformin is adjusted step by step to support the continuous and efficient proliferation of the cells; in the gradient maturation stage, the combination system of N-acetyl cysteine, TWS119 and DL-alpha tocopheryl acetate is introduced to help maintain the functional activity and antioxidant state of the cells in the later culture stage. The gamma delta T cells with a purity higher than 90% and an expansion multiple more than 100 times can be obtained by using the method to culture for 10 days, and the cells exhibit significant killing activity to tumor cells, are suitable for the adoptive immunotherapy of tumor and infectious diseases, and have good clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the phased and precise regulation of in vitro expansion and culture of highly active γδT cells. Background Technology

[0002] γδT cells, a subset of T lymphocytes with a unique antigen recognition mechanism, can recognize a variety of stress antigens and pathogen-associated molecules independently of MHC molecules, thus playing an important role in anti-tumor, antiviral, and antibacterial immune responses. In recent years, adoptive immunotherapy based on γδT cells has become one of the research hotspots in tumor biotherapy.

[0003] However, current in vitro expansion techniques for γδT cells still face several prominent challenges: First, the initial activation efficiency of cells is often low. Conventional methods rely on limited stimulation such as zoledronic acid combined with IL-2, which is insufficient to fully activate heterogeneous γδT cell populations, resulting in generally low early activation rates. Second, cell proliferation capacity is limited during expansion, and growth arrest or early senescence are common, usually leading to low expansion folds. Third, in the later stages of a long culture period, cells are prone to oxidative damage and functional decline, resulting in a significant decrease in cytotoxic activity. Finally, most existing methods require 12 to 14 days to obtain a sufficient number of functionally qualified cells, which is insufficient to meet the needs of rapid clinical preparation.

[0004] Therefore, there is an urgent need to develop a systematic culture method that can achieve efficient activation, stable expansion, high activity maintenance, and shortened cycle of γδT cells. Summary of the Invention

[0005] The purpose of this invention is to provide a staged and precise in vitro expansion and culture method for highly active γδT cells. This method significantly improves the activation level, expansion efficiency and functional persistence of γδT cells, while simultaneously greatly shortening the preparation cycle.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for staged and precise regulation of in vitro expansion and culture of highly active γδT cells, comprising the following steps: S1 Preparation of peripheral blood mononuclear cells (PBMCs): PBMCs were isolated from human peripheral blood; S2 Co-activation culture of γδT cells: PBMCs obtained in step S1 were seeded into activation medium for culture, which contained serum-free basal medium, zoledronic acid, IL-2, IL-15, IL-12, IL-21 and autologous plasma. Dynamic expansion culture of S3 γδT cells: After activation culture, the culture was replaced with expansion medium, which contained serum-free basal medium, IL-2, IL-15, autologous plasma and metformin. S4 γδT cell gradient maturation optimization culture: During the expansion culture process, TWS119, DL-α-tocopherol acetate and N-acetylcysteine ​​were added to the culture system for induction culture. After the culture was completed, the cells were harvested to obtain highly active γδT cells.

[0007] Preferably, the preparation of PBMCs in step S1 includes: centrifuging heparin-anticoagulated human peripheral blood, collecting the upper plasma layer and inactivating it for later use; diluting the centrifuged blood cell pellet with physiological saline, then slowly adding it to the upper layer of lymphocyte separation medium for density gradient centrifugation, collecting the white membrane layer cells, washing and resuspending them to obtain PBMCs.

[0008] Preferably, the plasma inactivation treatment includes the following steps: sequentially subjecting the collected plasma to 56°C water bath inactivation, centrifugation to obtain the supernatant, -20°C freezing treatment, and centrifugation again to obtain the supernatant.

[0009] Preferably, the concentrations of each component in the activation culture medium in step S2 are: zoledronic acid 5 μmol / L, IL-2 100 IU / mL, IL-15 10 ng / mL, IL-12 5 ng / mL, IL-21 8 ng / mL, and 5% autologous plasma; the cell density of the culture is 2 × 10⁻⁶ cells / mL. 6 Cells / mL, culture time is 3 days.

[0010] Preferably, the concentrations of each component in the amplification culture medium in step S3 are: IL-2 100 IU / mL, IL-15 10 ng / mL, 5% autologous plasma and metformin.

[0011] Preferably, the metformin is added as follows: on day 3 of amplification, it is added to a final concentration of 3 mM, and on day 5, it is added to a final concentration of 5 mM.

[0012] Preferably, the addition in step S4 is performed on the 7th day of culture from the date of inoculation in step S2, by adding TWS119 to the culture system at a final concentration of 1 μM, DL-α-tocopherol acetate at a final concentration of 1 mg / L, and N-acetylcysteine ​​at a final concentration of 0.5 mM; on the 9th day of culture, TWS119 and DL-α-tocopherol acetate are added again, and the final concentration after the addition is the same as the final concentration after the addition on the 7th day; the culture is continued until the 10th day, and the cells are collected.

[0013] The present invention also provides highly active γδT cells prepared by the method described above.

[0014] The present invention also provides the application of highly active γδT cells prepared by the above-described method in the preparation of a kit for in vitro killing of tumor cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Through innovative strategies such as multi-dimensional factor synergistic activation, staged metabolic optimization, and small molecule gradient induction, this method significantly improves the activation level, expansion efficiency, and functional persistence of γδT cells, while simultaneously achieving a substantial reduction in the preparation cycle.

[0016] The resulting cell products have high purity, strong cytotoxic activity, and good batch stability, demonstrating good potential for standardized preparation and clinical application value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a curve showing the fold expansion of γδT cells.

[0019] Figure 2 For γδT cell culture surface markers (CD3) + CDTCRγδ + The images show the results, with the left image representing day 0 of the culture and the right image representing day 10 of the culture.

[0020] Figure 3 This is a graph showing the killing effect of γδT cells on different tumor cell lines.

[0021] Figure 4 To synergistically activate different combinations of cytokines during the CD69 phase of γδT cells + A graph showing the impact of expression.

[0022] Figure 5 A graph illustrating the effect of different metformin addition strategies on cell proliferation. Detailed Implementation

[0023] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0024] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0025] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0026] Example 1 A staged and precise method for the in vitro expansion and culture of highly active γδT cells includes the following steps: 1. Preparation of PBMCs: A. Take 30mL~50mL of heparin-anticoagulated human peripheral blood into a 50mL centrifuge tube (each centrifuge tube contains approximately 30mL of blood), centrifuge at room temperature (800g) for 8 minutes.

[0027] B. After centrifugation, transfer the upper plasma layer to a new 50mL centrifuge tube using a 10mL pipette, and inactivate it in a 56℃ water bath for 30 minutes (shaking halfway through the 15-minute interval). After the plasma is inactivated, it will appear turbid. Centrifuge at 900g for 10 minutes. Take the supernatant and freeze it at -20℃ for 15 minutes (shaking well halfway through). After freezing, centrifuge again at 900g for 10 minutes, and store the supernatant at 4℃ for later use.

[0028] C. Add an equal volume of physiological saline to each centrifuge tube and mix thoroughly by pipetting. Slowly add the diluted peripheral blood to the top layer of the prepared lymphocyte separation medium (15 mL / tube) in each centrifuge tube, adding 25 mL to each tube.

[0029] D. Balance the centrifuge adapter, centrifuge at 800 g for 20 minutes, increase the pressure by 6 and decrease it by 1, with the temperature at 20℃.

[0030] E. After centrifugation, transfer the white membrane cells from the middle of the centrifuge tube to a new centrifuge tube, add physiological saline to 50 mL, invert to mix, and centrifuge at 500 g for 10 minutes at room temperature.

[0031] F. After centrifugation, discard the supernatant, resuspend the cells in serum-free culture medium, take a 0.1 mL sample and count the cells for later use.

[0032] 2. Co-activation culture of γδT cells: An activation medium was prepared by adding zoledronic acid (5 μmol / L), IL-2 (100 IU / mL), IL-15 (10 ng / mL), IL-12 (5 ng / mL), IL-21 (8 ng / mL), and 5% autologous plasma to serum-free medium. The concentration of the above PBMCs was adjusted to 2 × 10⁻⁶ cells / mL using an activation medium. 6 Cells / ml, initial culture of 10 mL, seeded into T75 cell culture flask, mixed well, and incubated for 3 days in an incubator at 37℃, 5% CO2, and saturated humidity.

[0033] 3. Dynamic Expansion Culture of γδT Cells: After 3 days of cell culture in step 2, aspirate the old culture medium and add 3 mL of new medium to the flask to prevent drying. Centrifuge at 350 g for 5 minutes, discard the supernatant, add 7 mL of expansion medium, resuspend, and transfer to a T75 flask. The expansion medium consists of serum-free medium supplemented with 100 IU / mL IL-2, 10 ng / mL IL-15, 5% autologous plasma, and metformin. Observe cell growth every 2 days, take a small number of cells for counting, and add expansion medium as needed (transfer to a larger culture flask or bag depending on the medium volume) to maintain a cell density of 2 × 10⁶ cells / mL. 6 Approximately one cell per mL.

[0034] 4. Optimized culture for gradient maturation of γδT cells: On day 7 of culture, add TWS119 to a final concentration of 1 μM, DL-α-tocopherol acetate to a final concentration of 1 mg / L, and N-acetylcysteine ​​to a final concentration of 0.5 mM. On day 9, add TWS119 and DL-α-tocopherol acetate again (at the same concentration as on day 7). Continue culturing until day 10, then collect the cells to obtain highly active γδT cells.

[0035] Example 2: Plotting Cell Expansion Growth Curves The γδT cell suspension cultured for days 3-10 of Example 1 was selected, and the number of viable cells was determined using a cell counter. A cell expansion and growth curve was plotted with culture time on the horizontal axis and expansion fold on the vertical axis. Figure 1 .

[0036] The formula for calculating the amplification factor is as follows: Expansion factor = Total number of viable cells on day n ÷ Initial total number of viable cells inoculated.

[0037] according to Figure 1 The test results show that the γδT cells cultured using the method provided by this invention have the advantages of rapid proliferation and high cell yield. By day 10 of culture, the cell expansion factor exceeded 100 times, which can fully meet the needs of clinical application.

[0038] Example 3 Immunophenotyping Detection Immunophenotyping was performed using flow cytometry to analyze the purity of γδT cells. The specific steps are as follows: γδT cells cultured on days 0 and 10 were collected, filtered, and then the cell density was adjusted to 1×10⁻⁶ cells / cells with buffer. 6 Cells were centrifuged at 350 g for 5 minutes and washed twice with PBS buffer. The supernatant was discarded, and the cells were resuspended in PBS. The cell suspension was aliquoted into EP tubes at 100 μL / tube, and 5 μL of CD3-FITC / TCR γδ-PE double staining antibody was added to each tube. After thorough mixing, the cells were stained. The cells were incubated at room temperature in the dark for 30 minutes, followed by centrifugation at 350 g for 5 minutes and washing once with PBS. Finally, the cells were resuspended in 200 μL of PBS, and the cell phenotype was analyzed by flow cytometry, and CD3+ was calculated. + TCRγδ + The percentage of double-positive cells (i.e., γδT cells).

[0039] The results are as follows Figure 2 As shown, the left image represents day 0 of culture, with a γδT cell positivity rate of 2.40%; the right image represents day 10 of culture, with the positivity rate increasing to 93.39%. These data confirm that the γδT cells cultured using the method of this invention have high purity.

[0040] Example 4: Testing the killing efficiency against tumor cells A549, K562, SK-OV-3, and HepG2 cell lines in logarithmic growth phase were selected as target cells, and the cell suspension density was adjusted to 1×10⁻⁶. 5 Cells / mL, 100 μL per well, were seeded into a 96-well culture plate. γδT cells harvested on day 10 were centrifuged and washed, and the cell suspension density was adjusted to 1×10⁻⁶. 6 Cells / mL, 100 μL per well, seeded into a 96-well plate, with an effector-to-target ratio of 10:1, and 4 replicates per group. Seeding method as follows: ① Untreated control (a value): Tumor cell culture medium (100 μL) + γδT culture medium (100 μL); ② Background control (b-value): Tumor cells (100 μL) + γδT culture medium (100 μL); ③Total LDH (c value): Tumor cells (100 μL) + γδT culture medium (100 μL); ④Total LDH background (d value): Tumor cell culture medium (100 μL) + γδT culture medium (100 μL); ⑤ Sample (e value): γδT cells (100 μL) + tumor cells (100 μL); ⑥ Sample background (f value): γδT cells (100 μL) + tumor cell culture medium (100 μL); After inoculating the cells, incubate them at 37°C with 5% CO2 for 5 hours and 45 minutes. Add 20 μL of lysis buffer to each well of the total LDH group and the total LDH background group, and add 20 μL of PBS to each of the other groups. Continue incubation for another 15 minutes. After incubation, centrifuge the 96-well plate at 400 g for 5 minutes. Transfer 100 μL of supernatant from each well to a new 96-well plate, add 100 μL of detection solution to each well, and incubate at room temperature for approximately 30 minutes. Measure the absorbance using a microplate reader at dual wavelengths of 450 nm / 630 nm. Calculate the cytotoxic activity using the following formula: Kill rate = (e value - f value - (a value - b value)) / [(c value - d value) - (a value - b value)] × 100%.

[0041] The test results are shown in Table 1 and Figure 3 As shown.

[0042] Table 1. Detection of the killing efficiency of γδT cells against different tumor cells.

[0043] Depend on Figure 3 The test results in Table 1 show that the γδT cells cultured to day 10 can achieve a killing rate of over 85% against four different types of tumor cells, confirming that the γδT cells prepared by the method provided in this invention have broad-spectrum and highly efficient tumor-killing activity.

[0044] Example 5: Effect of IL-21 on cell activation rate To clarify the role of IL-21 in the co-activation phase of γδT cells, this invention includes a control experiment to compare the effects of different IL-21 addition methods on CD69 in γδT cells. + The impact of the positive rate.

[0045] The activation medium of the experimental group (group 5-A) contained IL-21, which is consistent with the technical solution of Example 1 of the present invention; the activation medium of the control group (group 5-B) did not contain IL-21, and the remaining components were completely the same as the technical solution of Example 1 of the present invention.

[0046] On day 3 of culture, CD69 levels in γδT cells were detected. +The cell percentage was detected using the same method as in Example 3. The results are as follows: Figure 4 As shown in Table 2.

[0047] Table 2. Effects of different IL-21 addition methods on CD69 in γδT cells. + Impact of activation rate

[0048] The detection results showed that CD69 in γδT cells of group 5-A + The expression level was significantly higher than that of the 5-B group. The above data confirm that the addition of IL-21 plays a key role in significantly improving the early activation rate of γδT cells.

[0049] Example 6: Effect of metformin addition strategy on proliferation effect To clarify the effect of metformin on cell proliferation, this invention sets up a control experiment to compare the effects of different metformin addition strategies on cell proliferation.

[0050] The experimental group (6-A group) was added in stages, that is, metformin 3 mM was added on the 3rd day of culture and then increased to 5 mM on the 5th day of culture, which is consistent with the technical solution of Example 1; the control group (6-B group) had a final metformin concentration of 5 mM throughout the 3rd and 5th days of culture.

[0051] Cell expansion was assessed on day 7 of culture, using the same method as in Example 2. Results are as follows: Figure 5 As shown in Table 3.

[0052] The formula is: Amplification factor = Total number of viable cells on day 7 ÷ Total number of viable cells on day 3.

[0053] Table 3. Effects of different metformin addition strategies on cell proliferation.

[0054] The test results showed that the cell proliferation rate in group 6-A was significantly higher than that in group 6-B. These data confirm that the staged regulatory strategy of metformin can more effectively promote cell proliferation.

[0055] Example 7: Effects of the mature-stage antioxidant system on cellular reactive oxygen species (ROS) levels and cytotoxic activity. To clarify the effects of mature antioxidant systems on cellular reactive oxygen species (ROS) levels and cytotoxic activity, this invention sets up a control experiment to compare the effects of different combinations of antioxidant systems on cellular reactive oxygen species (ROS) levels and cytotoxic activity.

[0056] The experimental group (7-A group) was exactly the same as in Example 1; the control group (7-B group) was supplemented with only N-acetylcysteine; and the control group (7-C group) was completely free of N-acetylcysteine, TWS119 and DL-α-tocopherol acetate.

[0057] 7.1 Detection of cellular reactive oxygen species (ROS) levels On day 10, γδT cells from the experimental group (group 7-A), control group (group 7-B), and control group (group 7-C) were collected, washed twice with pre-cooled PBS buffer, centrifuged at 350 g for 5 minutes, and the supernatant was discarded. The cells were resuspended in serum-free medium and the cell density was adjusted to 1×10⁻⁶. 6 Quantity / mL, for later use.

[0058] Add 100 μL of cell suspension with adjusted density to a flow cytometer. According to the ROS detection kit instructions, add DCFH-DA probe solution to a final concentration of 10 μM. After gently mixing, incubate at 37°C in a 5% CO2 incubator in the dark for 20 minutes, gently inverting and mixing every 5 minutes to ensure the probe is fully loaded.

[0059] After incubation, add 2 mL of pre-cooled PBS buffer to stop staining, centrifuge at 350 g for 5 minutes, and discard the supernatant; repeat the washing step twice to completely remove free probes that have not entered the cells.

[0060] Resuspension assay: Cells were resuspended in 300 μL of pre-chilled PBS buffer, and the fluorescence intensity of the cells was detected by flow cytometry (excitation wavelength 488 nm, emission wavelength 525 nm); 1×10⁻⁶ cells were analyzed per sample. 4 The relative level of intracellular ROS was reflected by the mean fluorescence intensity of individual cells.

[0061] The mean fluorescence intensity (MFI) of cells in each group was calculated. Using the MFI of the control group (7-C group) as a baseline, the relative ROS levels of the experimental group (7-A group) and the control group (7-B group) were calculated using the following formula: Relative ROS level = 7-A / B group MFI ÷ 7-C group MFI × 100%.

[0062] The results are shown in Table 4.

[0063] Table 4. Effects of different combinations of antioxidant systems on cellular reactive oxygen species (ROS) levels and cytotoxic activity.

[0064] The test results showed that the relative ROS level of the 7-A group cells was significantly lower than that of the 7-B / C group, and the killing efficiency of the 7-A group cells was significantly higher than that of the 7-B / C group cells. The above data confirm that the composite antioxidant system of the present invention can significantly reduce the level of oxidative stress and effectively maintain the high killing activity of cells.

[0065] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for staged and precise regulation of in vitro expansion and culture of highly active γδT cells, characterized in that, Includes the following steps: S1 Preparation of peripheral blood mononuclear cells (PBMCs): PBMCs were isolated from human peripheral blood; S2 Co-activation culture of γδT cells: PBMCs obtained in step S1 were seeded into activation medium for culture, which contained serum-free basal medium, zoledronic acid, IL-2, IL-15, IL-12, IL-21 and autologous plasma. Dynamic expansion culture of S3 γδT cells: After activation culture, the culture was replaced with expansion medium, which contained serum-free basal medium, IL-2, IL-15, autologous plasma and metformin. S4 γδT cell gradient maturation optimization culture: During the expansion culture process, TWS119, DL-α-tocopherol acetate and N-acetylcysteine ​​were added to the culture system for induction culture. After the culture was completed, the cells were harvested to obtain highly active γδT cells.

2. The method according to claim 1, characterized in that, The preparation of PBMCs in step S1 includes: centrifuging heparin-anticoagulated human peripheral blood, collecting the upper plasma layer and inactivating it for later use; diluting the centrifuged blood cell pellet with physiological saline, then slowly adding it to the upper layer of lymphocyte separation medium for density gradient centrifugation, collecting the white membrane layer cells, washing and resuspending them to obtain PBMCs.

3. The method according to claim 2, characterized in that, The plasma inactivation process includes the following steps: sequentially subjecting the collected plasma to a 56°C water bath for inactivation, centrifuging to obtain the supernatant, freezing at -20°C, and centrifuging again to obtain the supernatant.

4. The method according to claim 1, characterized in that, The concentrations of each component in the activation culture medium described in step S2 are: zoledronic acid 5 μmol / L, IL-2 100 IU / mL, IL-15 10 ng / mL, IL-12 5 ng / mL, IL-21 8 ng / mL, and 5% autologous plasma; the cell density of the culture is 2 × 10⁻⁶ cells / mL. 6 Cells / mL, culture time is 3 days.

5. The method according to claim 1, characterized in that, The concentrations of each component in the amplification culture medium described in step S3 are: IL-2 100 IU / mL, IL-15 10 ng / mL, 5% autologous plasma and metformin.

6. The method according to claim 5, characterized in that, The metformin was added as follows: on day 3 of amplification, it was added to a final concentration of 3 mM, and on day 5, it was added to a final concentration of 5 mM.

7. The method according to claim 1, characterized in that, The addition described in step S4 is performed on day 7 of culture, starting from the inoculation date in step S2. A final concentration of 1 μM TWS119, a final concentration of 1 mg / L DL-α-tocopherol acetate, and a final concentration of 0.5 mM N-acetylcysteine ​​are added to the culture system. On day 9 of culture, TWS119 and DL-α-tocopherol acetate are added again, with the final concentrations after the addition being the same as those after the addition on day 7. Culture continues until day 10, at which point cells are collected.

8. Highly active γδT cells prepared by the method according to any one of claims 1-7.

9. The use of highly active γδT cells prepared by the method according to any one of claims 1-7 in the preparation of a kit for in vitro killing of tumor cells.

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