A culture method for promoting activation and expansion of gd t cells

Abstract

The present application relates to the field of cell biology, specifically will be by adding phosphoantigen (HMBPP), IL-2 and other factors, specific activation of gamma delta T cells, and Daudi lymphoma cell feeder layer co-culture, promote the expansion of gamma delta T cells, at the same time, promote the memory phenotype and survival of gamma delta T cells, and improve the cytotoxicity and function of gamma delta T cells. The method simultaneously induces peripheral blood mononuclear cells to become gamma delta T cells and activates gamma delta T cells, enhances immune signal transduction, and significantly improves the activation efficiency and expansion capacity. The results show that the proliferation of gamma delta T cells is 742.62 times in 14 days, which is significantly higher than that of conventional CIK culture; CD3 + cell ratio reaches 90.15%, which is better than 75.29% of CIK. Thus, a short cycle, high yield, and good purity gamma delta T cell preparation process is formed, which embodies the creativity of multi-factor synergy and feeder layer co-culture, and brings significant beneficial effects.

Description

Technical Field

[0001] This invention relates to the field of cell biology, specifically to a culture method that specifically activates γδT cells by adding factors such as HMBPP and IL-2, and then co-culturing them with Daudi lymphoma cell feeder layers. This promotes the expansion of γδT cells, enhances their memory phenotype and survival, and improves their cytotoxicity and function. It is a culture method that significantly improves the activation and expansion efficiency of γδT cells. Background Technology

[0002] With the deepening and development of cell biology and clinical medical technologies, research on the killing of tumor cells by immune cells has also gradually deepened. However, because tumor cells have the ability to evade the surveillance of the immune system, promoting and activating the signal transduction of immune cells, their recognition of tumor cells, and their killing effects are of great clinical significance for the development and clinical application of tumor treatment and immune cell therapy.

[0003] "γδT" is short for γδT cells, a unique and powerful sentinel and rapid-response force in the immune system. They operate independently of the MHL (mevaleosome-dependent lymphocytes), residing at key gateways in the body (mucous membranes and tissues). They rapidly detect danger signals from cellular abnormalities and take immediate action, playing an indispensable role in fighting infection, fighting tumors, and maintaining tissue homeostasis. γδT cells are a unique and important subset of T lymphocytes. The vast majority of T cells (approximately 95%) are αβT cells, whose TCRs consist of α and β chains. They recognize protein fragments (peptides) presented by the major histocompatibility complex (MHC) molecules. γδT cell killing is highly specific. Their TCRs can directly sense phosphate antigens produced by metabolic abnormalities within tumor cells (upregulation of the mevalonate pathway). This allows them to more reliably distinguish between cancer cells and normal cells. γδT cells are a homogeneous and unique subset of T cells. They possess characteristics of both T cells and NK cells, enabling them to specifically recognize and kill stress cells (such as tumor cells) in an MHC-unrestricted manner. Their killing is more "precise and efficient".

[0004] HMBPP (full name: (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate) inhibits the mevalonate pathway, leading to the accumulation of intracellular IPP (isopentenyl pyrophosphate). As a phosphate antigen, HMBPP specifically activates Vγ9Vδ2 T cells (the most prevalent γδ T cell subtype in the human body) and causes them to proliferate rapidly. IL-2 is a key cytokine for maintaining proliferation and survival.

[0005] There is a major subset of γδ T cells in the human body whose T cell receptors are composed of Vγ9 and Vδ2 chains (called Vγ9Vδ2 T cells). These cells can directly recognize HMBPP, but this recognition process is not the conventional "antigen peptide-MHC" mechanism. Recognition mechanism: HMBPP recognition depends on the BTN3A1 (CD277) molecule. The widely accepted model is that HMBPP binds to the intracellular domain of the BTN3A1 molecule. This binding leads to a conformational change in BTN3A1, which then transmits its signal extracellularly. Clusters of BTN3A1 molecules on the cell surface are then recognized by the TCR of Vγ9Vδ2 T cells, ultimately triggering strong T cell activation. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to specifically activate γδT cells by adding factors such as HMBPP and IL-2, and co-culturing them with Daudi lymphoma cell feeder layers. This promotes γδT cell proliferation, enhances the memory phenotype and survival of γδT cells, and improves their cytotoxicity and function.

[0007] The specific technical solution is as follows: A method for promoting the activation and expansion of γδT cells, characterized by comprising the following steps: Step 1, Monocyte isolation: Peripheral blood samples were collected and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation. The supernatant was discarded and cell pellet was obtained. The second step is antibody coating: anti-CD3 antibody and anti-TCRγδ antibody are mixed at concentrations of 0.5-5 μg / mL and 1-10 μg / mL, diluted with sterile PBS, added to a culture flask, and incubated overnight at 4°C; the coating solution is discarded, the mixture is washed 3 times with sterile PBS, and blocked with PBS solution containing 1-2% human serum albumin (HSA) for 1-2 hours, and the blocking solution is discarded; The third step, cell seeding and initial activation: The cell pellet from the first step was resuspended in TCM-199 medium at 30%-40% of the blood sample volume, and seeded into the culture flasks treated in the second step. Phosphoantigen HMBPP (final concentration 2.5 μM), IL-2 (final concentration 500 IU / mL), and IFN-γ (final concentration 10 ng / mL) were added, and the cells were cultured at 37°C in a 5% CO2 incubator. On the second day of culture, anti-CD3 antibody (CD3 AK, final concentration 50 ng / mL) and epidermal growth factor (EGF, final concentration 20 ng / mL) were added. Step 4, feeder cell irradiation treatment: Collect Daudi cells in the logarithmic growth phase and adjust the concentration to 20 × 10⁻⁶. 6cells / mL, inactivated by 80 Gy γ-ray irradiation, and the supernatant was discarded by centrifugation; Step 5, co-culture expansion: On day 4 of culture, add 5 × 10⁵ irradiated Daudi cells prepared in step 4 to the culture flask. 6 One sample, along with autologous peripheral blood serum and an equal volume of TCM-199 medium (containing IL-2 at a final concentration of 500 IU / mL), were cultured for another day; on day 5, twice the volume of the blood sample was added to TCM-199 medium. Step 6, Large-scale expansion: On day 7 of culture, transfer the cells to a sterile cell culture bag, add TCM-199 medium to a final volume of 15-18 times the volume of the blood sample, and simultaneously add IL-2 (final concentration 300 IU / mL), IL-15 (final concentration 200 IU / mL), IL-18 (final concentration 100 IU / mL), and IL-21 (final concentration 100 IU / mL), and continue culturing; Step 7, Cell Harvesting: On day 21 of culture, collect the cells, centrifuge and discard the supernatant to obtain expanded γδT cells.

[0008] This method provides a complete technical solution for high-rate expansion of high-purity γδT cells. Its core advantage lies in the integration of multiple activation and expansion mechanisms: 1) Specific initial activation: By coating the surface of γδ T cells with anti-TCRγδ antibody, the specific receptors are directly targeted, and combined with the phosphate antigen HMBPP (which mimics pathogen infection signals) and CD3 antibody (which provides T cell receptor co-stimulatory signals), efficient and specific activation of γδ T cell subsets is achieved. This avoids the large-scale expansion of αβ T cells caused by the traditional PHA / IL-2 protocol and significantly improves the purity of the γδ T cells in the initial culture. 2) Dynamic cytokine regulation: In the early stage, high concentration of IL-2 (500 IU / mL) promotes cells to enter the proliferation cycle rapidly. In the middle stage, IFN-γ is introduced to enhance the anti-tumor activity of cells. In the later stage, a low concentration of multi-factor combination of IL-2 (300 IU / mL) in the culture bag, together with IL-15 (maintaining memory phenotype), IL-18 (promoting IFN-γ secretion), and IL-21 (enhancing cytotoxicity), effectively prevents activation-induced cell death (AICD) and terminal differentiation induced by high concentration of IL-2, and maintains the long-term survival and functional activity of expanded cells. 3) Feeder cell co-culture: Irradiation-inactivated Daudi cells (Burkitt lymphoma cell line) not only serve as a physical scaffold to promote cell-to-cell contact, but their surface-expressed stress antigens (such as MICA / B) and secreted unknown factors can further specifically activate γδT cells. At the same time, autologous peripheral blood serum provides personalized growth factors and nutrients, significantly increasing the expansion rate. 4) Large-scale culture system: The transition from culture flasks to breathable culture bags, combined with a stepwise increase in culture medium volume, solves the expansion bottleneck caused by nutrient depletion and metabolic waste accumulation in traditional culture systems, achieving stable production from laboratory scale to clinical therapeutic levels. The final obtained γδT cell population has high purity (CD3+). + TCRγδ + With double positivity ≥90%, high amplification fold (≥500-fold), strong cytotoxicity (efficiently kills tumor cells such as Daudi), and high cytokine secretion capacity (IFN-γ, TNF-α), it fully meets the dual requirements of adoptive immunotherapy for cell quantity and quality.

[0009] The density gradient centrifugation described in the first step of this invention uses lymphocyte separation medium and centrifugation conditions of 2000 rpm for 20 min. These centrifugation conditions are optimized standard parameters based on the density differences of various components in human peripheral blood (monocyte density approximately 1.075-1.090 g / mL, granulocyte / erythrocyte ratio >1.090 g / mL, plasma <1.025 g / mL). The combination of centrifugation force and time of 2000 rpm for 20 min ensures that PBMCs (lymphocytes and monocytes) fully migrate to the separation medium interface during centrifugation to form a clear white film layer, while minimizing contamination from erythrocytes and granulocytes. Compared to other centrifugation conditions, these parameters guarantee the highest recovery rate (typically >85%) and optimal viability (viable cell rate >95%) of PBMCs, providing a sufficient number of healthy seed cells for subsequent expansion and avoiding subsequent culture contamination or low expansion efficiency due to incomplete separation.

[0010] In the second step of this invention, the coating concentrations of the anti-CD3 antibody and the anti-TCRγδ antibody are 2 μg / mL and 5 μg / mL, respectively. This combination of concentrations is an optimized ratio that has been verified through numerous experiments. The coating concentration of 5 μg / mL of the anti-TCRγδ antibody ensures sufficient antibody density on the bottom surface of the culture flask, maximizing the capture of γδT cells in PBMCs and improving the specificity of initial inoculation. The concentration of 2 μg / mL of the anti-CD3 antibody provides a moderate co-stimulatory signal, which can synergistically promote the activation and proliferation of γδT cells with the TCRγδ signal, while avoiding the risk of non-specific T cell activation or cytokine storm caused by excessive CD3 stimulation. This concentration can initiate γδT cell proliferation faster than using the anti-TCRγδ antibody alone, and has higher γδT cell selectivity than using the anti-CD3 antibody alone. It is the key to achieving high purity and high efficiency of initial activation.

[0011] The TCM-199 culture medium described in the third step of this invention contains 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. This TCM-199 medium is rich in amino acids, vitamins, nucleic acid precursors, and other nutrients, meeting the metabolic needs of rapid cell proliferation. The 10% FBS provides essential growth factors (such as IGF and FGF), hormones (such as insulin), and lipids, while adhesion proteins (such as fibronectin) in the serum help cells adhere to the culture medium. The 1% penicillin-streptomycin (typically 100 U / mL penicillin and 100 μg / mL streptomycin) effectively inhibits contamination by Gram-positive and Gram-negative bacteria, ensuring a sterile culture environment for several weeks. This culture medium formulation achieves an optimal balance between supporting cell growth and preventing contamination, providing a fundamental guarantee for successful cell expansion.

[0012] The culture bag described in step six of this invention is made of ethylene-vinyl acetate copolymer (EVA) and has a 0.2 μm breathable membrane. This 0.2 μm breathable membrane allows for the free exchange of O2 and CO2, maintaining a stable pH (approximately 7.2-7.4) and dissolved oxygen level within the culture bag. This is crucial for high-density cell culture, preventing the shift of cell metabolism to lactic acid fermentation and decreased cell viability due to hypoxia. Simultaneously, the 0.2 μm pore size effectively blocks the invasion of bacteria and fungi, maintaining the sterility of the culture system. Compared to traditional rigid culture flasks, EVA culture bags offer a larger surface area to volume ratio, facilitating uniform cell distribution and nutrient diffusion. They are also easy to handle on a large scale (e.g., heat sealing, centrifugation harvesting), making them an ideal carrier for clinical-grade expansion of γδT cells.

[0013] CD3 in the γδT cell population described in step seven of this invention + TCRγδ + The proportion of double-positive cells is not less than 90%, and the cell amplification fold is not less than 500-fold. The CD3+... + TCRγδ + A double-positive cell ratio ≥90% indicates that this method has extremely high selectivity for γδT cells, with very few confounding αβT cells, NK cells, or other immune cells in the amplification products. This is crucial for adoptive immunotherapy, maximizing the anti-tumor effect of γδT cells while reducing off-target toxicity and the risk of graft-versus-host disease (GvHD). An amplification fold ≥500-fold means that therapeutic doses (10) can be obtained from a small amount of peripheral blood (e.g., 50 mL). 9 -10¹ 0 This technology utilizes γδT cells, overcoming the bottleneck of low γδT cell percentage (1-5%) in the human body and the difficulty in obtaining sufficient quantities for treatment. The combination of high purity and high quantity ensures the effectiveness and feasibility of the treatment.

[0014] This invention relates to the application of a novel phosphoantigen (HMBPP) as an activator, aiming to enhance the activity and proliferation capacity of γδ T cells, thereby increasing their potential efficacy in tumor immunotherapy. Its beneficial effects are mainly reflected in the following aspects: First, HMBPP possesses unique biological characteristics, allowing it to be taken up by various cell types, including osteoclasts, monocytes, and tumor cells. After HMBPP uptake, these cells effectively inhibit farnesyl pyrophosphate synthase (FPPS), leading to the accumulation of the important metabolic intermediate isopentenyl pyrophosphate (IPP). Due to the blockade of the mevalonate pathway, the intracellular concentration of IPP significantly increases, providing the necessary endogenous signaling for the activation of γδT cells.

[0015] Secondly, accumulated IPP can be presented to γδT cells through cellular release or other mechanisms. Studies have found that the T cell receptor (TCR) of the major Vγ9Vδ2 T cell subset can specifically recognize IPP, thereby generating a strong activation signal. This recognition not only promotes the rapid activation and proliferation of γδT cells, but also enhances their ability to kill tumor cells and secrete cytokines (such as interferon-γ).

[0016] This invention also proposes a unique cell culture protocol that further promotes the maturation and expansion of γδT cells by adding CD3 antibody (CD3AK), interleukin-2 (IL-2), interleukin-15 (IL-15), interleukin-18 (IL-18), and interleukin-21 (IL-21) during the culture process. Studies co-culturing with Daudi lymphoma cells have shown that this culture protocol significantly improves the maturation rate of γδT cells, while simultaneously increasing the γδT cell expansion efficiency by 742.62 times.

[0017] Furthermore, this protocol combines multiple activation and expansion mechanisms, achieving specific activation through anti-TCRγδ antibody and phosphate antigen HMBPP, significantly improving the purity of γδT cells. Co-culturing with dynamically regulated cytokines and irradiated-inactivated Daudi cells not only enhances cell viability and function but also facilitates large-scale culture, ultimately resulting in a γδT cell population with high purity, high expansion rate, and strong cytotoxicity.

[0018] In summary, this invention proposes an innovative method for activating and expanding γδT cells through the application of the phosphate antigen HMBPP, demonstrating its potential effectiveness in enhancing immune responses and anti-tumor effects, and has broad clinical application prospects and market value. Attached Figure Description

[0019] Figure 1 These are images of γδT cells under a microscope after induction; Figure 2This refers to the fold increase in the number of γδT cells compared to conventional CIK cells; Figure 3 It is the CD3 of γδT cells and conventional CIK cells + Proportional situation. Detailed Implementation

[0020] A method for promoting the activation and expansion of γδT cells, characterized by the following culture steps: The first step is the isolation of mononuclear cells. A quantitative peripheral blood sample was placed into a 50 mL centrifuge tube and centrifuged, discarding the residue at the bottom. The obtained blood cells were diluted with physiological saline and slowly added to a 50 mL centrifuge tube containing 20%-30% of the blood sample volume of lymphocyte separation medium. After centrifugation, the supernatant was discarded. The white blood cell layer was slowly transferred to a new centrifuge tube, and 5 mL of physiological saline was added to the centrifuge tube containing the white blood cell layer. The mixture was stirred well to obtain a white blood cell dilution. The final white blood cell dilution was 70%-80% of the blood sample volume. The white blood cell dilution was centrifuged and the supernatant was discarded. The cells were resuspended in 5%-10% of the blood sample volume of physiological saline, and then centrifuged again with 70%-80% of the blood sample volume of physiological saline. The supernatant was discarded to obtain the cell pellet. The second step is antibody coating. Coat new culture flasks with anti-CD3 and anti-TCRγδ antibodies. The commonly used coating concentrations for anti-CD3 antibody are 0.5–5 μg / mL, and for anti-TCRγδ antibody, 1–10 μg / mL. Take a sterile test tube and add the calculated volume of sterile PBS. Add the calculated volumes of both antibodies to the test tube and gently pipette to mix. Add the mixed antibody solution to the culture flask, ensuring the liquid evenly covers the entire bottom. Cap the flask and gently shake to distribute the solution evenly. Incubate the culture flask overnight (16–18 hours) at 4°C. The next day, carefully aspirate the antibody coating solution from the culture flask in a biosafety cabinet. Add sterile PBS or physiological saline (at least 2–3 times the original coating solution volume) to the flask and gently shake to rinse the bottom. Discard the rinsing solution. Repeat this rinsing step 2–3 times to thoroughly wash away unbound free antibodies. Add the prepared blocking solution (PBS / culture medium containing 1-2% HSA or FBS) to the culture flask, the volume of which should be the same as or slightly more than the volume of the coating solution. Incubate at room temperature for at least 30 minutes to 2 hours. Discard the blocking solution. Rinse the bottom of the flask 1-2 times with sterile PBS, aspirating any remaining liquid.

[0021] The third step is to inoculate the culture flasks. Resuspend the cell pellet in 5%-10% of the blood sample volume of TCM-199 medium, then add 25%-35% of the blood sample volume of TCM-199 medium again, mix well, and place in a T175 culture flask. Wash the centrifuge tube with 30%-60% of the blood sample volume of TCM-199 medium, and pour the washing solution into a T175 culture flask. Add phosphoantigen (HMBPP), IL-2, and IFN-γ to the T175 culture flask (final concentration of HMBPP: 2.5 μM, final concentration of IL-2: 500 IU / mL, final concentration of IFN-γ: 10 ng / mL), and incubate the flask in a CO2 incubator. On the second day, add CD3AK and EGF to the culture flask. Step 4: Irradiation treatment of feeder cells Collect Daudi cells in the logarithmic growth phase into centrifuge tubes. Centrifuge at 1000 rpm for 5 minutes, discarding the old culture medium. Resuspend the cells in PBS or serum-free medium and count them. Adjust the cell concentration to 20 × 10⁶ cells / mL using complete culture medium. 6 High-density (cells / mL) irradiation. Transfer the cells to the irradiator operator. The key irradiation parameter is the dose. Place the centrifuge tube containing the cell suspension in the center of the irradiator's sample chamber to ensure uniform dose distribution. The radiation dose is 80 Gy. After irradiation, remove the cell tube. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant containing free radicals.

[0022] Step 5: Co-culture of feeder cells On the 4th day, [the sample was] containing 5×10 [units of something]. 6 Activated γδT cells were added to a culture flask containing Daudi cells, along with peripheral blood serum obtained when monocytes were acquired and an equal volume of fresh TCM-199 medium (containing IL-2 at a final concentration of 500 IU / mL). The flask was then placed in a 5% CO2 incubator for incubation. On day 5, twice the volume of the blood sample in TCM-199 medium was added to the culture flask.

[0023] Step 6: Inoculate the culture bag After culturing in culture flasks for 7 days, cells were transferred to culture bags. The culture flasks were poured into culture bags, and the flasks were washed with TCM-199 medium. The washings were then poured into the culture bags, and TCM-199 medium was added to the bags until the final volume was 15-18 times the volume of the blood sample. Simultaneously, IL-2, IL-15, IL-18, and IL-21 were added (IL-2 final concentration 300 IU / mL, IL-15 final concentration 200 IU / mL, IL-18 final concentration 100 IU / mL, IL-21 final concentration 100 IU / mL). The distal end of the inlet tube of the culture bag was sealed with a heat sealer. The culture bags were then placed in a CO2 incubator for further culture. Step 7: Collect cells After culturing in the culture bag for 14 days, the cell culture bag is sterilized with 70%-75% alcohol. The cells in the cell culture bag are mixed and placed in a biosafety cabinet. The scissors and the outlet tube on the culture bag are sterilized with povidone-iodine. The outlet tube of the culture bag is cut open, the cell culture medium is centrifuged, and the supernatant is discarded to obtain γδT cells.

[0024] This invention combines the addition of phosphoantigen HMBPP, CD3 agonist antibody (CD3 AK), and IL-2, IL-15, IL-18, and IL-21 to the culture system, and co-cultures it with a feeder layer of Daudi lymphoma cells to construct a high-intensity co-stimulatory environment. This method simultaneously induces peripheral blood mononuclear cells to become γδT cells and activates γδT cells, enhancing immune signal transduction and significantly improving activation efficiency and proliferation capacity. Results showed that γδT cells proliferated 742.62-fold after 14 days, significantly higher than conventional CIK culture; CD3... + The cell ratio reached 90.15%, which is superior to CIK's 75.29%. This led to the formation of a short-cycle, high-yield, and high-purity γδT cell preparation process, demonstrating the creativity of multi-factor synergy and feeder layer co-culture, and bringing significant beneficial effects.

Claims

1. A culture method for promoting the activation and expansion of γδT cells, characterized in that, Includes the following steps: Step 1, Monocyte isolation: Peripheral blood samples were collected and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation. The supernatant was discarded and cell pellet was obtained. The second step is antibody coating: anti-CD3 antibody and anti-TCRγδ antibody are mixed at concentrations of 0.5-5 μg / mL and 1-10 μg / mL, diluted with sterile PBS, added to a culture flask, and incubated overnight at 4°C; the coating solution is discarded, the mixture is washed 3 times with sterile PBS, and blocked with PBS solution containing 1-2% human serum albumin (HSA) for 1-2 hours, and the blocking solution is discarded; The third step, cell seeding and initial activation: The cell pellet from the first step was resuspended in TCM-199 medium at 30%-40% of the blood volume, and seeded into the culture flasks treated in the second step. A final concentration of 2.5 μM phosphate antigen HMBPP, a final concentration of 500 IU / mL IL-2, and a final concentration of 10 ng / mL IFN-γ were added, and the cells were cultured at 37℃ in a 5% CO2 incubator. On the second day of culture, anti-CD3 antibody and epidermal growth factor EGF were added. Step 4, feeder cell irradiation treatment: Collect Daudi cells in the logarithmic growth phase and adjust the concentration to 20 × 10⁻⁶. 6 cells / mL, after being inactivated by 80 Gy γ-ray irradiation, the supernatant was discarded by centrifugation; Step 5, co-culture expansion: On day 4 of culture, add 5 × 10⁵ irradiated Daudi cells prepared in step 4 to the culture flask. 6 One sample, along with autologous peripheral blood serum and an equal volume of TCM-199 medium, was cultured for another day. The TCM-199 medium contained IL-2L at a final concentration of 500 IU / m³. On day 5, twice the volume of TCM-199 medium was added to the blood sample. Step 6, Large-scale expansion: On day 7 of culture, transfer the cells to a sterile cell culture bag, add TCM-199 medium to a final volume of 15-18 times the blood sample volume, and simultaneously add IL-2 at a final concentration of 300 IU / mL, IL-15 at a final concentration of 200 IU / mL, IL-18 at a final concentration of 100 IU / mL, and IL-21 at a final concentration of 100 IU / mL, and continue culturing; Step 7, Cell Harvesting: On day 21 of culture, collect the cells, centrifuge and discard the supernatant to obtain expanded γδT cells.

2. The method according to claim 1, characterized in that, The density gradient centrifugation in the first step uses lymphocyte separation medium and centrifugation conditions of 2000 rpm for 20 min.

3. The method according to claim 1, characterized in that, The coating concentrations of the anti-CD3 antibody and the anti-TCRγδ antibody mentioned in the second step are 2 μg / mL and 5 μg / mL, respectively.

4. The method according to claim 1, characterized in that, The TCM-199 culture medium described in step three contains 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

5. The method according to claim 1, characterized in that, The culture bag mentioned in step six is ​​made of ethylene-vinyl acetate copolymer (EVA) and has a 0.2 μm breathable membrane.

6. The method according to claim 1, characterized in that, CD3 in the γδT cell population described in step seven + TCRγδ + The proportion of double-positive cells is not less than 90%, and the cell amplification factor is not less than 500 times.

7. The method according to claim 1, characterized in that, The final concentration of CD3AK was 50 ng / mL, and the final concentration of epidermal growth factor (EGF) was 20 ng / mL.

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