Culture medium composition for culturing GDT cells and culture method

Abstract

The invention discloses a culture medium composition for culturing GDT cells and a culture method, and belongs to the field of biological medicine. Aiming at the problems of low in-vitro amplification efficiency and poor function of the existing GDT cells, the invention provides the culture medium composition for culturing the GDT cells, the culture medium composition comprises an induction culture medium and a proliferation culture medium, the induction culture medium comprises a basic culture medium and an activating agent, and the activating agent comprises IL-7 and paclitaxel; the proliferation culture medium comprises a basic culture medium, IL-18 and a metabolism regulator. An STAT5 pathway is activated through IL-7, and amplification of the Vdelta2 subgroup is specifically promoted; on the other hand, through the cooperation of the IL-7 and the IL-18, the IL-18 activates the expression of the NKG2D, and the IL-18 and the IL-7 jointly enhance the cytotoxicity; efficient amplification and function enhancement of the GDT cells are achieved; the killing rate of PD-L1 + tumor cells is larger than 80%, the motility rate after cryopreservation recovery is larger than 90%, the function maintenance rate is larger than 85%, and the cell line is suitable for tumor immunotherapy, infectious disease treatment and cell drug development.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a culture medium combination and a culture method for culturing GDT cells. Background Art

[0002] GDT cells possess non-MHC-restricted killing capabilities, but their in vitro expansion faces challenges such as low efficiency (<50-fold with traditional methods), imbalanced subpopulation ratios (e.g., Vδ1 / Vδ2), and metabolic limitations. Existing technologies often rely on a single cytokine, IL-2 or IL-15, for in vitro expansion, which can easily lead to Treg cell contamination (>10%) and cell exhaustion. They also fail to integrate the synergistic effects of multiple cytokines, failing to balance proliferation and functional differentiation, resulting in a single signaling pathway. Furthermore, they lack targeted design for specific expansion of the Vδ2 subpopulation, leading to insufficient subpopulation regulation. Furthermore, they fail to optimize the dynamic balance between glycolysis and OXPHOS, resulting in impaired mitochondrial function and a lack of metabolic support.

[0003] Corresponding improvements have also been made to address the above-mentioned problems, such as Chinese patent publication number CN114736857A, published on July 12, 2022. This patent discloses an induction culture medium, a culture medium combination, and a culture method for culturing new γδT cells, wherein the induction culture medium components include a γδT cell basal culture medium and an inducer AR A014418. The culture medium combination includes an induction culture medium and an expansion culture medium; the induction culture medium components include a γδT cell basal culture medium and an inducer AR A014418; the expansion culture medium components include a γδT cell basal culture medium, IL 7, IL 15, IL 18, IL 21, and AB plasma. The shortcoming of this patent is that there will be antagonistic contradictions in the joint action of multiple cytokines in the expansion culture medium. For example, the combination of IL-18 and IL-12 may induce fatal pulmonary edema, and IL 15 at supraphysiological concentrations will induce the expansion of regulatory T cells (Tregs), which in turn inhibits the function of effector T cells.

[0004] Another example is Chinese patent application number CN113684179A, published on November 23, 2021, which discloses a cell expansion culture medium, a combination product and a culture method for γδT. The γδT cell expansion culture medium includes a basal culture medium and additional components, and the additional components include: 5% to 15% (v / v) FBS, IL2 200IU / mL to 800IU / mL, IL211ng / mL to 20ng / mL and coenzyme Q10 5μmol / L to 20μmol / L. The shortcoming of this patent is that FBS is a complex mixture, and its growth factor, hormone and metabolite content varies significantly due to individual fetal cattle, collection season and processing method, and its stability is easily affected. Summary of the Invention

[0005] 1. Problems to be solved

[0006] To address the slow in vitro expansion efficiency and poor functionality of existing GDT cells, the present invention provides a culture medium combination and culture method for culturing GDT cells. IL-7 in the culture medium combination specifically activates and expands the Vδ2 subset, while IL-18 activates NKG2D expression. These two synergistically inhibit FoxP3 expression, reduce Treg contamination, and enhance cytotoxicity, achieving efficient expansion and functional enhancement of GDT cells. Furthermore, the GDT cells cultured using this method meet clinical standards, balancing safety and functionality.

[0007] 2. Technical solution

[0008] To solve the above problems, the present invention adopts the following technical solutions.

[0009] A culture medium combination for culturing GDT cells comprises an induction culture medium and a proliferation culture medium, wherein the induction culture medium comprises a basal culture medium and an activator, wherein the activator comprises IL-7 and paclitaxel; and the proliferation culture medium comprises a basal culture medium, IL-18 and a metabolic regulator.

[0010] Furthermore, the concentration of IL-7 in the induction medium is 10-20 ng / mL, the concentration of paclitaxel is 15-25 μg / mL; the concentration of IL-18 in the proliferation medium is 8-12 ng / mL, the concentration of the metabolic regulator is 3-7 μM, and the metabolic regulator is metformin.

[0011] Furthermore, it also includes a functional enhancement culture medium, which includes a basal culture medium and a compound stimulation factor, and the compound stimulation factor is a mixture of IL-7 and IL-18.

[0012] Furthermore, the concentration of IL-7 in the composite stimulating factor is 5-10 ng / mL, and the concentration of IL-18 is 5-10 ng / mL.

[0013] Furthermore, the basal culture medium in the induction culture medium and the proliferation culture medium both includes a mixture of TAKARA-GT-T551 and RPMI-1640, and a metabolic support agent.

[0014] Furthermore, the volume ratio of TAKARA-GT-T551 to RPMI-1640 in the mixture of TAKARA-GT-T551 and RPMI-1640 is 1:2; and the metabolic support agents are sodium pyruvate and α-ketoglutaric acid.

[0015] A method for culturing GDT cells comprises culturing GDT cells using any of the above-described culture medium combinations for culturing GDT cells.

[0016] Furthermore, when the proliferation stage culture is carried out using the proliferation medium, the metabolic regulator is added at set intervals.

[0017] 3. Beneficial effects

[0018] (1) The present invention is creative in that, on the one hand, by adding cytokine IL-7 to the induction medium, IL-7 activates the STAT5 pathway, specifically promotes the expansion of the Vδ2 subpopulation, and significantly increases the proportion of Vδ2 subpopulation expansion. At the same time, paclitaxel assists in enhancing the TCRγδ signal response. On the other hand, by synergizing IL-7 in the induction medium and IL-18 in the proliferation medium, IL-18 activates NKG2D expression through the NF-κB pathway and enhances cytotoxicity together with IL-7. Ultimately, efficient expansion and functional enhancement of GDT cells can be achieved, breaking through the bottleneck of traditional technologies, and is suitable for tumor immunotherapy, infectious disease treatment and cell drug development.

[0019] (2) The present invention strictly controls the concentrations of IL-7 and IL-18, which can ensure the efficient expansion of Vδ2T cells while accurately inhibiting Treg contamination, thereby providing high-purity cell resources for subsequent use; because IL-7 directs the expansion of the Vδ2 subpopulation, IL-18 enhances NKG2D expression; through the synergy of the two, it is possible to downregulate FoxP3 expression, and the residual Treg ratio is less than 0.5%, thereby inhibiting Treg cell contamination; therefore, abnormal IL-7 concentrations will lead to nonspecific expansion, cell depletion, or insufficient expansion of Vδ2 subpopulation cells; abnormal IL-18 concentrations will lead to insufficient NKG2D expression or excessive cytotoxicity, killing normal cells or causing imbalance in the culture system;

[0020] (3) The present invention also adds a composite stimulatory factor of IL-7 and IL-18 as the core of the functional enhancement culture medium, which activates IFN-γ secretion through the STAT5 / NF-κB dual pathway to stimulate and enhance killing activity; the setting of the functional enhancement culture medium can avoid cell loss caused by signal interruption during staged culture, and at the same time, through the continuous high secretion of IFN-γ and the synergistic effect of effector molecules, maximize the killing activity, and ultimately achieve a killing rate of PD-L1+ tumor cells of >80%, a viability rate of >90% after cryopreservation and recovery, and a functional maintenance of >85%;

[0021] (4) The basal medium in the functionalized culture medium of the present invention does not use serum, thus avoiding the uncertainty of growth factor, hormone and metabolite content caused by the use of animal serum due to differences in individual, collection season and processing method; at the same time, the metabolic support agent is used to support the dynamic balance of glycolysis-OXPHOS;

[0022] (5) The core of the GDT cell culture method of the present invention is to activate and expand the Vδ2 subpopulation specifically through IL-7 in the culture medium combination, and activate NKG2D expression through IL-18. The two synergistically inhibit FoxP3 expression, reduce Treg contamination, and enhance cytotoxicity; so that the cultured GDT cells meet clinical standards: taking into account both safety and functionality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of cell purity in Example 1 of the present application;

[0024] Figure 2 This is a schematic diagram of the cell killing ability in Example 1 of the present application. DETAILED DESCRIPTION

[0025] To better understand the core of this application, the applicant now briefly summarizes the existing technology: Existing GDT cell in vitro expansion usually uses different cytokines for culture, which has the following limitations:

[0026] L-4 or IL-9: Th2 polarization: IL-4 promotes Th2 cell differentiation, secretes IL-5 and IL-13, inhibits the cytotoxicity of GDT cells (killing rate decreases by 40%), skin barrier destruction: IL-9 upregulates keratin degradation enzymes, resulting in reduced keratinocyte adhesion, which is associated with skin autoimmune diseases such as psoriasis;

[0027] IL-6 or IL-12: Excessive inflammation: IL-6 induces the STAT3 pathway, triggering a cytokine storm (e.g., CRP > 20 mg / L), increasing the risk of CRS in CAR-T therapy; metabolic imbalance: Although IL-12 combined with TGF-β can promote Th1 differentiation, it does not integrate metabolic regulators (e.g., α-ketoglutarate), leading to glycolysis / OXPHOS imbalance (apoptosis rate > 20%);

[0028] IL-21 or IL-18: Single function: IL-21 alone can enhance B cell antibody secretion, but cannot specifically expand the Vδ2 subset (accounting for <50%); Mitochondrial damage: IL-18 alone leads to ROS accumulation and mitochondrial membrane potential maintenance rate of <60%;

[0029] IGF-1 or IL-33: Promote tumor microenvironment: IGF-1 promotes tumor cell proliferation, IL-33 induces Th2 type immunosuppression, and weakens the anti-tumor function of GDT cells.

[0030] The present invention is further described below with reference to specific examples and accompanying drawings. This application effectively addresses the core drawbacks of single cytokines through the coordinated design and optimized concentrations of IL-7 and IL-18. The GDT cells in this application are γδT cells, and the experiments performed are all routine experiments in the field.

[0031] Example 1

[0032] Specifically, a method for culturing GDT cells comprises the following steps:

[0033] S1: Sorting and activation: Peripheral blood mononuclear cells (purity > 95%) were sorted using TCRγδ magnetic beads, with a cell number of 5 × 10;

[0034] S2: Induction culture was performed for a fixed time in an induction medium comprising the following components: a mixture of TAKARA-GT551 and RPMI-1640 (volume ratio 1:2), sodium pyruvate (50 μM), α-ketoglutarate (100 μM), IL-7 (15 ng / mL), and paclitaxel (20 μg / mL); IL-7 was used in the induction medium to activate the STAT5 pathway and initiate proliferation of the Vδ2 subset. It should be noted that TAKARA-GT551 is a serum-free culture medium designed specifically for human lymphocytes, dendritic cells (DCs), and natural killer cells (NK cells); RPMI-1640 is a basal cell culture medium developed by the Roswell Park Memorial Institute in the United States, and the code name "1640" represents its formula number. Both culture media have clear compositions and are conventional culture media in the art, and therefore will not be described in detail in this example.

[0035] S3: Cells were cultured for a fixed period of time in a proliferation medium containing a mixture of GT-T551 and RPMI-1640 (volume ratio 1:2), sodium pyruvate (50 μM), α-ketoglutarate (100 μM), IL-18 (10 ng / mL), and metformin (5 μM). IL-18 enhances cytotoxicity via the NF-κB pathway and synergizes with IL-7 to enhance cytotoxicity via the NF-κB / STAT5 dual pathway. During the proliferation phase, 5 μM metformin was added every 48 hours to inhibit the mTORC1 pathway and maintain mitochondrial autophagy.

[0036] S4: Functional enhancement culture was performed for a fixed time in a functional enhancement medium containing the following components: a mixture of GT-T551 and RPMI-1640 (volume ratio 1:2), sodium pyruvate (50 μM), α-ketoglutarate (100 μM); IL-7 (10 ng / mL), IL-18 (8 ng / mL); combined stimulation with IL-7 and IL-18 was continued for 48 h, activating IFN-γ secretion through the STAT5 / NF-κB dual pathway, resulting in IFN-γ secretion >1200 pg / mL.

[0037] The quality inspection of the GDT cells cultured above yielded the following results: flow cytometry verification showed that the proportion of the Vδ2 subpopulation was >78%, the LDH killing rate was >85% (efficacy-target ratio 10:1), and the killing rate for A549 lung cancer cells was 92% (efficacy-target ratio 10:1, traditional method 55%); Treg residue was <0.5%; mitochondrial membrane potential (ΔΨm) maintenance rate was >90%, and the apoptosis rate was <5%; GDT cells met the following clinical standards: safety: no Treg contamination (CD4+FoxP3+ <0.5%), avoiding the risk of graft-versus-host disease (GVHD); serum-free process, endotoxin level <0.1EU / mL (in compliance with FDA / EMA standards); functionality: killing rate for PD-L1+ tumor cells was >80%; viability after cryopreservation and resuscitation was >90%, and functional maintenance was >85%.

[0038] Its cell purity and cell killing ability are as follows Figure 1 and Figure 2 As shown in the figure, Donor1, Donor2 and Donor3 are all from frozen peripheral blood. Figure 1 and Figure 2 It can be seen that the GDT cells cultured using the culture medium combination of the present application have high purity and strong lethality.

[0039] At the same time, the inventors of the present application conducted the following experiment: GDT cells cultured using the medium combination of the present application were compared with GDT cells cultured using the above-mentioned existing medium using other cytokines alone. It is noted that except for the different culture medium used, all other variables in the entire culture conditions remained the same; the results are shown in Table 1 below:

[0040] Table 1 Performance test diagram

[0041]

[0042] GDT cells cultured using the medium combination of the present application were compared with GDT cells cultured using the above-mentioned existing combination of culture media with other cytokines. Note that, except for the different culture media used, all other variables remained the same throughout the culture conditions. To verify the inventive advantages of the combination of IL-7 and IL-18, the following control group experiment was set up (n=4, effector-target ratio 10:1, culture period 14 days): The results are as follows:

[0043] IL-2+IL-15IL-2:100IU / mL-Vδ2 subpopulation proportion <40%

[0044] - Residual Treg ratio > 10%

[0045] -IFN-γ secretion <300pg / mL IL-2 promotes Treg differentiation (FoxP3↑), and supraphysiological concentrations of IL-15 induce cell exhaustion (PD-1 expression >40%)

[0046] IL-21+IL-12IL-21:10ng / mL-IFN-γ secretion <800pg / mL

[0047] -Mitochondrial membrane potential maintenance rate <60%

[0048] - Apoptosis rate > 20% IL-12 induces ROS accumulation, IL-21 does not integrate metabolic support (ATP < 1.5 nM / 10 cells)

[0049] IL-4+IL-9IL-4:20ng / mL-Vδ1 subpopulation proportion>60%

[0050] - Kill rate decreased by 40%

[0051] - Increased activity of skin barrier-related enzymes: IL-4 drives Th2 polarization (inhibits cytotoxicity), and IL-9 upregulates keratin-degrading enzymes (associated with skin autoimmune diseases)

[0052] IL-6+TGF-βIL-6:50ng / mL-CRP>20mg / L (inflammation risk)

[0053] - Glycolysis / OXPHOS imbalance (apoptosis rate > 25%), IL-6 activation of the STAT3 pathway (cytokine storm), and TGF-β-induced immunosuppressive microenvironment

[0054] IL-18 alone: 15 ng / mL - NKG2D expression > 80%

[0055] -ROS accumulation increased 3 times

[0056] - Mitochondrial membrane potential maintenance rate <50% lacks IL-7 synergy, cannot inhibit Treg differentiation, and high concentration of IL-18 leads to metabolic stress;

[0057] IL-7+IL-18 (this application) IL-7: 15ng / mL - Vδ2 subpopulation proportion > 75%

[0058] - Treg residual <0.5%

[0059] -IFN-γ>1200pg / mL synergistically activates the STAT5 / NF-κB pathway, inhibits FoxP3 expression, and balances glycolysis-OXPHOS metabolism

[0060] Based on the above experiments, the following conclusions can be drawn:

[0061] IL-2+IL-15: Treg contamination is serious and cannot meet clinical safety requirements;

[0062] IL-21+IL-12: Metabolic imbalance leads to mitochondrial damage and poor functional sustainability;

[0063] IL-4+IL-9: Subpopulation regulation failure and risk of autoimmune disease;

[0064] IL-6+TGF-β: Dual risks of inflammation and immunosuppression, not suitable for tumor treatment;

[0065] IL-18 alone: Although it enhances NKG2D expression, it lacks Vδ2-specific amplification and exhibits significant metabolic toxicity.

[0066] IL-7+IL-18 (this application): the only combination that achieves directed expansion of the Vδ2 subset, Treg suppression, metabolic balance and high cytotoxicity, verifying its irreplaceable nature.

[0067] Example 2

[0068] Specifically, in this embodiment, a method for culturing GDT cells includes the following steps:

[0069] S1: Sorting and activation: Peripheral blood mononuclear cells (purity > 95%) were sorted using TCRγδ magnetic beads, with a cell number of 5 × 10;

[0070] S2: Induction culture was performed for a fixed time in an induction medium containing the following components: a mixture of GT-T551 and RPMI-1640 (volume ratio 1:2), sodium pyruvate (50 μM), α-ketoglutarate (100 μM), IL-7 (15 ng / mL), and paclitaxel (20 μg / mL); IL-7 activated the STAT5 pathway in the induction medium, initiating proliferation of the Vδ2 subpopulation;

[0071] S3: Cells were cultured for a fixed period of time in a proliferation medium containing the following components: a mixture of GT-T551 and RPMI-1640 (volume ratio 1:2), sodium pyruvate (50 μM), α-ketoglutarate (100 μM); IL-18 (10 ng / mL), and metformin (5 μM). In the proliferation medium, IL-18 enhanced cytotoxicity via the NF-κB pathway and synergized with IL-7 to enhance cytotoxicity via the NF-κB / STAT5 dual pathway. During the proliferation culture phase, 5 μM metformin was added every 48 h to inhibit the mTORC1 pathway and maintain mitochondrial autophagy.

[0072] The test results are as follows: Vδ2 subpopulation proportion: 75% (better than the 50% of the traditional method); PD-L1+ tumor cell killing rate: 70% (significantly higher than the 55% of the traditional method); survival rate after cryopreservation and resuscitation: 85% (the traditional method is 70%); function maintenance (IFN-γ secretion): 800pg / mL (the traditional method is 300pg / mL); Treg residual ratio: 1.2% (the traditional method is >10%); while in Example 1, the Vδ2 subpopulation proportion: 78% (an increase of 3% compared with Example 2); PD-L1+ tumor cell killing rate: 85% (an increase of 15% compared with Example 2); survival rate after cryopreservation and resuscitation: 90% (an increase of 5% compared with Example 2); function maintenance (IFN-γ secretion): 1200pg / mL (an increase of 50% compared with Example 2); Treg residual ratio: 0.5% (a decrease of 0.7% compared with Example 2).

[0073] Comparative Example 1

[0074] The results were basically the same as those in Example 1, except that the induction medium in step S2 did not contain IL-7, and the rest remained the same. The test results were as follows: the proportion of the Vδ2 subpopulation dropped to 45%, the GDT subpopulation dominated, and the anti-solid tumor activity decreased by 50%; the residual Treg ratio increased to 8%, PD-1 expression was greater than 30%, leading to cell exhaustion; IFN-γ secretion was less than 300 pg / mL, and the apoptosis rate was greater than 25%; the ATP level was less than 1.5 nM / 10 cells, and the ROS accumulation increased by 3 times.

[0075] Comparative Example 2

[0076] The procedure was essentially the same as Example 1, except that two control induction culture media were provided in step S2. The first control induction culture medium contained 5 ng / mL of IL-7, while the second control induction culture medium contained 25 ng / mL of IL-7. The remaining control culture media components were identical to those in the induction culture medium in Example 1. The remaining steps remained the same as in Example 1. The following results were obtained:

[0077] Group 1: The Vδ2 subpopulation accounted for only 35%, the expansion fold was <200-fold, and the residual Treg ratio was >5%;

[0078] Group 2: The Vδ2 subpopulation accounted for 50%, but the cells were significantly exhausted (PD-1 expression >40%) and the apoptosis rate was >20%;

[0079] Example 1: The Vδ2 subpopulation accounts for 78%, the expansion factor is 600 times, and the residual Treg is <0.5%.

[0080] From the above results, it can be seen that as the concentration of IL-7 continues to increase, it can effectively and specifically promote the expansion of the Vδ2 subpopulation, causing the proportion of Vδ2 subpopulation expansion to increase significantly; however, too high a concentration will inhibit the expansion of the Vδ2 subpopulation and increase the risk of cell exhaustion; therefore, determining the appropriate IL-7 concentration can not only achieve a significant increase in the proportion of Vδ2 subpopulation expansion but also effectively inhibit Treg cell contamination.

[0081] Comparative Example 3

[0082] The method was essentially the same as Example 1, except that, in step S4, four control functional enhancement culture media were set up. The first control functional enhancement culture medium contained 12 ng / mL of IL-7 and 8 ng / mL of IL-18; the second control functional enhancement culture medium contained 3 ng / mL of IL-7 and 8 ng / mL of IL-18; the third control functional enhancement culture medium contained 8 ng / mL of IL-7 and 15 ng / mL of IL-18; and the fourth control functional enhancement culture medium contained 8 ng / mL of IL-7 and 3 ng / mL of IL-18. The remaining components of the functional enhancement culture medium were the same as those in Example 1. The remaining steps were the same as in Example 1, and the results were as follows:

[0083] Group 1: IFN-γ secretion <500 pg / mL, killing rate decreased to 50%;

[0084] Group 2: IFN-γ secretion <500 pg / mL, killing rate decreased to 60%;

[0085] Group 3: excessive cytotoxicity (killing normal cells>30%), apoptosis rate>25%;

[0086] Group 4: insufficient NKG2D expression (<50%), killing rate <50%;

[0087] Example 1: IFN-γ secretion > 1200 pg / mL, killing rate > 80%.

[0088] From the above results, it can be seen that abnormal IL-7 concentration will lead to nonspecific expansion, cell exhaustion or insufficient expansion of Vδ2 subpopulation cells; abnormal IL-18 concentration will lead to insufficient NKG2D expression or excessive cytotoxicity, killing normal cells or causing imbalance in the culture system; therefore, the appropriate concentrations of IL-7 and IL-18 in the functional enhanced culture medium can maximize the killing activity through the continuous high secretion of IFN-γ and the synergistic effect of effector molecules.

[0089] The examples described in the present invention are merely descriptions of the preferred embodiments of the present invention and are not intended to limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various modifications and improvements made to the technical solutions of the present invention by engineers and technicians in this field should fall within the scope of protection of the present invention.

Claims

1. A culture medium composition for culturing GDT cells, characterized in that: The invention comprises an induction medium and a proliferation medium. The induction medium comprises a basic medium and an activator, and the activator comprises IL-7 and paclitaxel; and the proliferation medium comprises a basic medium, IL-18 and a metabolic regulator.

2. The culture medium combination for culturing GDT cells according to claim 1, characterized in that: The concentration of IL-7 in the induction medium is 10-20 ng / mL, and the concentration of paclitaxel is 15-25 μg / mL; the concentration of IL-18 in the proliferation medium is 8-12 ng / mL, and the concentration of the metabolic regulator is 3-7 μM, and the metabolic regulator is metformin.

3. A culture medium combination for culturing GDT cells according to claim 1 or 2, characterized in that: It also includes a functional enhancement culture medium, which includes a basic culture medium and a compound stimulation factor, and the compound stimulation factor is a mixture of IL-7 and IL-18.

4. The culture medium combination for culturing GDT cells according to claim 3, characterized in that: The concentration of IL-7 in the composite stimulating factor is 5-10 ng / mL, and the concentration of IL-18 is 5-10 ng / mL.

5. The culture medium combination for culturing GDT cells according to claim 1, characterized in that: The basal culture medium in the induction culture medium and the proliferation culture medium both includes a mixture of TAKARA-GT-T551 and RPMI-1640, and a metabolic support agent.

6. The culture medium combination for culturing GDT cells according to claim 5, characterized in that: The volume ratio of TAKARA-GT-T551 to RPMI-1640 in the mixed solution of TAKARA-GT-T551 and RPMI-1640 is 1:2; the metabolic support agents are sodium pyruvate and α-ketoglutaric acid.

7. A method for culturing GDT cells, characterized in that: The GDT cells are cultured using the culture medium combination for culturing GDT cells according to any one of claims 1 to 6.

8. The method for culturing GDT cells according to claim 7, characterized in that: During the proliferation phase of culture using a proliferation medium, metabolic regulators are added at set intervals.

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