Gamma delta T cell serum-free additive culture medium composition with definite components, and application and product of gamma delta T cell serum-free additive culture medium composition

Abstract

The invention belongs to the technical field of cytobiology and immune cell therapy, and particularly relates to a gamma delta T cell serum-free additive culture medium composition with definite components and application and a product thereof. The serum-additive-free culture medium composition comprises a serum-additive-free culture medium A, a serum-free culture addition reagent B, a serum-additive-free culture medium C, a serum-additive-free culture medium D and a serum-additive-free culture medium E, the main components comprise ethanolamine, sodium selenite, recombinant human albumin, IL-15 and IL-2. According to the gamma delta T cell serum-free additive culture medium composition provided by the invention, the components are clear, the serum-free culture additives without animal-derived components can avoid potential safety hazards caused by the animal-derived components, the amplification efficiency is improved, the cell high activity is maintained, the GMP standard is met, and the gamma delta T cell serum-free additive culture medium composition is suitable for large-scale production and clinical transformation.

Description

Technical Field

[0001] This invention belongs to the field of cell biology and immune cell therapy technology, specifically relating to a serum-free culture medium composition for γδT cells with clearly defined components, its application, and products. Background Technology

[0002] Based on the different TCR chains, T cells can be divided into αβT cells and γδT cells. Unlike αβT cells, the T cell receptor on the surface of γδT cells is composed of γ and δ chains. Although γδT cells only account for 1-10% of peripheral blood T cells, they play a crucial role in the immune response, especially as a bridge between innate and adaptive immunity, playing a key role in anti-tumor and anti-infective immunity. γδT cells can exert their tumor-killing effects by secreting various cytokines (such as IFN-γ and TNF-α) and chemokines; they can also induce tumor cell apoptosis by upregulating the expression of FasL and tumor necrosis factor-related apoptosis-inducing ligands; they can also induce cell apoptosis by releasing perforin and granzymes; and they can exert anti-tumor effects through their antigen presentation. Among γδT cells, Vγ9 and Vδ2 T cells have been studied the most, accounting for 50-75% of peripheral blood γδT cells. They can recognize and be activated by various ligands, such as non-peptide antigens and phosphate antigens, playing an important role in tumor immunity and immune regulation.

[0003] Traditional amplification systems often rely on animal serum (FBS or human platelet lysate, etc.) or high doses of IL-2 to maintain proliferation and activity, which presents three problems: (1) complex serum composition and large batch-to-batch differences lead to unstable amplification results; (2) animal / human-derived components bring potential pathogen and immunogenic risks, which are not conducive to clinical translation and release testing; (3) strong stimulation strategies such as high doses of IL-2 or TLR agonists are prone to cause cytokine storm risk and overactivation, affecting product safety and consistency.

[0004] Simultaneously, γδT cells undergo significant metabolic remodeling and increased oxidative stress 1–3 days after activation. If the antioxidant network and membrane phospholipid renewal are not synchronized, early apoptosis is commonly observed, followed by impaired proliferation and functional fatigue. Existing serum-free systems often neglect the dynamic ratio and timing of administration of ethanolamine (a membrane phospholipid precursor) and selenium (an antioxidant enzyme substrate). Furthermore, albumin, as a substitute for serum function, is often only considered as a carrier, while its functions of sustained nutrient release and metabolic homeostasis support are not systematically utilized. Therefore, there is an urgent need for a component-defined serum-free system that can balance expansion, purity, viability, and function under low IL-2 conditions, and can precisely regulate stress and metabolic balance within critical cellular windows through ratio and timing. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a serum-free culture medium composition for γδT cells with clearly defined components, as well as its applications and products.

[0006] In this invention, the term "defined" means that all components and concentrations are quantifiable and recordable, and that the product is free of serum and its derivatives. The term "xeno-free" means that the product contains no animal-derived components and uses recombinant human proteins. The term "final working concentration" refers to the concentration obtained after serum-free culture with added reagents to the basal medium and reaching the required operating conditions. In this invention, percentage concentration is expressed as a weight-to-volume percentage (w / v), and volume ratio is expressed as (v / v).

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a serum-free culture medium combination for γδT cells with clearly defined components, the serum-free culture medium combination comprising: serum-free culture medium A, serum-free culture additive B, serum-free culture medium C, serum-free culture medium D, and serum-free culture medium E; The serum-free supplement culture medium A consists of ethanolamine, sodium selenite, recombinant human albumin, IL-15, excipients, and basal culture medium; The serum-free culture additive B consists of ethanolamine, sodium selenite, and basal culture medium, wherein the molar ratio of ethanolamine to sodium selenite is 3.75-12:1; The serum-free culture medium C, serum-free culture medium D, and serum-free culture medium E are composed of ethanolamine, sodium selenite, recombinant human albumin, IL-15, IL-2, excipients, and basal culture medium; the IL-2 content in serum-free culture medium C is 350-450 IU / mL; the IL-2 content in serum-free culture medium D is 150-250 IU / mL; and the IL-2 content in serum-free culture medium E is 50-150 IU / mL.

[0008] Specifically, the serum-free culture medium A contains 100-300 nM ethanolamine, 20-60 nM sodium selenite, 2.5-7.5 mg / mL recombinant human albumin, and 6-10 ng / mL IL-15.

[0009] More specifically, the serum-free culture medium A contains 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290 or 290-300 nM ethanolamine.

[0010] Preferably, the serum-free culture medium A contains 200 nM ethanolamine.

[0011] More specifically, the serum-free culture medium A contains 20-30, 30-40, 40-50, or 50-60 nM sodium selenite.

[0012] Preferably, the serum-free supplement culture medium A contains 40 nM sodium selenite. More specifically, the serum-free culture medium A contains 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0 or 7.0-7.5 mg / mL of recombinant human albumin.

[0013] Preferably, the serum-free culture medium A contains 5 mg / mL recombinant human albumin.

[0014] More specifically, the serum-free culture medium A contains 6-7, 7-8, 8-9, or 9-10 ng / mL IL-15.

[0015] Preferably, the serum-free culture medium A contains 8 ng / mL IL-15.

[0016] Specifically, the molar ratio of ethanolamine to sodium selenite in the serum-free culture reagent B is 3.75-4.00:1, 4.00-4.25:1, 4.25-4.50:1, 4.50-4.75:1, 4.75-5.00:1, 5.00-5.25:1, 5.25-5.50:1, 5.50-5.75:1, 5.75-6.00:1, 6.00-6.25:1, 6.25-6.50:1, 6.50-6.75:1, 6.75-7.00:1, 7.00-7.25:1, 7.25-7.50:1, and 7.50-7.75:1. 7.75-8.00:1, 8.00-8.25:1, 8.25-8.50:1, 8.50-8.75:1, 8.75-9.00:1, 9.00-9.25:1, 9.25-9.50:1, 9.50-9.75:1, 9.75-10.00:1, 10.00-10.25:1, 10.25-10.50:1, 10.50-10.75:1, 10.75-11.00:1, 11.00-11.25:1, 11.25-11.50:1, 11.50-11.75:1 or 11.75-12.00:1.

[0017] Preferably, the molar ratio of ethanolamine to sodium selenite in the serum-free culture reagent B is 3.75-4.00:1, 4.00-4.25:1, 4.25-4.50:1, 4.50-4.75:1, 4.75-5.00:1, 5.00-5.25:1, 5.25-5.50:1, 5.50-5.75:1, 5.75-6.00:1, 6.00-6.25:1, 6.25-6.50:1, 6.50-6.75:1, 6.75-7.00:1, 7.00-7.25:1, or 7.25-7.50:1.

[0018] More preferably, the molar ratio of ethanolamine to sodium selenite in the serum-free culture reagent B is 5.75-6.00:1 or 6.00-6.25:1.

[0019] Specifically, the method of using the serum-free culture reagent B is to add it in three equal molar amounts, with the total target amount being 200-1200 nM ethanolamine and 40-160 nM sodium selenite.

[0020] More specifically, the total target amount of reagent B added for serum-free culture is 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100 or 1100-1200 nM of ethanolamine.

[0021] Preferably, the total target amount of reagent B added for serum-free culture is 600 nM ethanolamine.

[0022] More specifically, the total target amount of sodium selenite added to the serum-free culture reagent B is 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150 or 150-160 nM.

[0023] Preferably, the total target amount of reagent B added for serum-free culture is 100 nM sodium selenite.

[0024] Specifically, the serum-free culture reagent C contains 100-300 nM ethanolamine, 20-60 nM sodium selenite, 2.5-7.5 mg / mL recombinant human albumin, 6-10 ng / mL IL-15, and 350-450 IU / mL IL-2.

[0025] More specifically, the serum-free culture medium C contains 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290 or 290-300 nM ethanolamine.

[0026] Preferably, the serum-free culture medium C contains 200 nM ethanolamine.

[0027] More specifically, the serum-free culture medium C contains 20-30, 30-40, 40-50, or 50-60 nM sodium selenite.

[0028] Preferably, the serum-free culture medium C contains 40 nM sodium selenite. More specifically, the serum-free culture medium C contains 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0 or 7.0-7.5 mg / mL of recombinant human albumin.

[0029] Preferably, the serum-free culture medium C contains 5 mg / mL recombinant human albumin.

[0030] More specifically, the serum-free culture medium C contains 6-7, 7-8, 8-9, or 9-10 ng / mL IL-15.

[0031] Preferably, the serum-free culture medium C contains 8 ng / mL IL-15.

[0032] More specifically, the serum-free culture medium C contains 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440 or 440-450 IU / mL IL-2.

[0033] Preferably, the serum-free culture medium C contains 400 IU / mL IL-2.

[0034] Specifically, the serum-free culture medium D contains 100-300 nM ethanolamine, 20-60 nM sodium selenite, 2.5-7.5 mg / mL recombinant human albumin, 6-10 ng / mL IL-15, and 150-250 IU / mL IL-2.

[0035] Preferably, the serum-free culture medium D contains 200 nM ethanolamine.

[0036] Preferably, the serum-free culture medium D contains 40 nM sodium selenite. Preferably, the serum-free culture medium D contains 5 mg / mL recombinant human albumin.

[0037] Preferably, the serum-free culture medium D contains 8 ng / mL IL-15.

[0038] More specifically, the serum-free culture medium D contains 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240 or 240-250 IU / mL IL-2.

[0039] Preferably, the serum-free culture medium D contains 200 IU / mL IL-2.

[0040] Specifically, serum-free culture medium E contains 100-300 nM ethanolamine, 20-60 nM sodium selenite, 2.5-7.5 mg / mL recombinant human albumin, 6-10 ng / mL IL-15, and 50-150 IU / mL IL-2.

[0041] Preferably, the serum-free culture medium E contains 200 nM ethanolamine.

[0042] Preferably, the serum-free culture medium E contains 40 nM sodium selenite. Preferably, the serum-free culture medium E contains 5 mg / mL recombinant human albumin.

[0043] Preferably, the serum-free culture medium E contains 8 ng / mL IL-15.

[0044] More specifically, the serum-free culture medium E contains 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140 or 140-150 IU / mL IL-2.

[0045] Preferably, the serum-free culture medium D contains 100 IU / mL IL-2.

[0046] Specifically, the excipient combination includes: L-alanyl-L-glutamine, N-acetylcysteine, ascorbic acid, recombinant human transferrin, and recombinant human insulin.

[0047] More specifically, the serum-free culture medium composition contains L-alanyl-L-glutamine at a concentration of 2-6 mM, N-acetylcysteine ​​at a concentration of 0.5-1.5 mM, ascorbic acid at a concentration of 100-300 μM, recombinant human transferrin at a concentration of 2.5-7.5 mg / L, and recombinant human insulin at a concentration of 2.5-7.5 mg / L.

[0048] Preferably, the serum-free culture medium composition contains L-alanyl-L-glutamine at a concentration of 4 mM, N-acetylcysteine ​​at a concentration of 1 mM, ascorbic acid at a concentration of 200 μM, recombinant human transferrin at a concentration of 5 mg / L, and recombinant human insulin at a concentration of 5 mg / L.

[0049] Specifically, the basal culture medium includes any one or more of RPMI-1640 medium, IMDM medium, DMEM / F12 medium, and α-MEM medium.

[0050] Preferably, the basal culture medium is RPMI-1640 medium.

[0051] Specifically, the serum-free culture medium A, serum-free culture medium C, serum-free culture medium D and serum-free culture medium E have a pH of 7.2-7.6 and an osmotic pressure of 310-330 mOsm / kg.

[0052] Preferably, the serum-free culture medium A, serum-free culture medium C, serum-free culture medium D and serum-free culture medium E have a pH of 7.4 and an osmotic pressure of 320 mOsm / kg.

[0053] In another aspect, the present invention provides the use of the serum-free culture medium combination described in any of the above claims in the preparation of in vitro expanded γδT cell products.

[0054] In another aspect, the present invention provides a product for in vitro expansion of γδT cells, the product comprising the serum-free culture medium combination described in any of the preceding claims.

[0055] Preferably, the product includes any one or more of the following: γδT cell amplification preparation, γδT cell amplification kit, and γδT cell amplification chip.

[0056] Preferably, the product further includes any one or more of the following: cell activator, cell cryopreservation protectant, cell diluent, sterile culture consumables, and quality control reagents.

[0057] In another aspect, the present invention provides a method for in vitro expansion of γδT cells, the method comprising using the serum-free culture medium combination or product described in any of the preceding claims.

[0058] Specifically, the method includes the following steps: S1, Activation phase: PBMCs are seeded in serum-free medium A, and cell activators and cytokines are added to obtain the activation medium; S2. Segmented pulse supplementation stage: Add serum-free culture additive B to the activation medium. The method of using serum-free culture additive B is to add it in 3 equal molar amounts. The total target amount of adding is 200-1200 nM ethanolamine and 40-160 nM sodium selenite. S3, Maintenance growth stage: Discard the original culture medium and replace it with serum-free culture medium C, and culture for 3-4 days; Discard the original culture medium and replace it with serum-free culture medium D, and culture for 3-4 days; Discard the original culture medium and replace it with serum-free culture medium E, and culture for 3-4 days. S4. Harvesting stage: Harvest cells and wash them to obtain expanded γδT cells.

[0059] Preferably, the γδT cells prepared by the method are used for one or more of the following: prevention and / or treatment of tumor immunity, prevention and / or treatment of infectious diseases, and immune regulation.

[0060] In another aspect, the present invention provides γδT cells prepared by the method described in any of the above-mentioned methods.

[0061] In another aspect, the present invention provides the use of γδT cells prepared by the method described in any of the above-mentioned methods in the preparation of drugs for the prevention and / or treatment of tumor immunity, the prevention and / or treatment of infectious diseases, and immunomodulatory drugs.

[0062] In another aspect, the present invention provides a method for preventing and / or treating tumor immunity, preventing and / or treating infectious diseases, and immune regulation, wherein the method comprises using γδT cells prepared by the method described in any of the above-mentioned methods.

[0063] The beneficial effects of this invention are as follows: The serum-free culture medium combination for γδT cells provided by this invention has a clearly defined composition and is free of animal-derived components. This avoids the safety risks associated with animal-derived components, improves amplification efficiency, maintains high cell viability, and meets GMP standards, making it suitable for large-scale production and clinical translation. Attached Figure Description

[0064] Figure 1 Comparison of the effects of different molar ratios of ethanolamine to selenite on γδT cell expansion; (A) Vδ2 + Purity; (B) Key proportion group Vδ2 + (C) Purity difference analysis; (D) Amplification fold; (E) Comparison of amplification folds in key proportion groups; (N) Cell viability; Data are expressed as Mean ± SD (n=3); Statistical analysis was performed using t-tests or one-way ANOVA; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0065] Figure 2 The effects of different methods of adding ethanolamine and sodium selenite on the expansion and purity of γδT cells; (A) γδT cell expansion fold; (B) γδT cell Vδ2+ Proportion (purity, %); (C) γδT cell viability; data are expressed as Mean ± SD (n=3); statistical analysis was performed using t-tests or one-way ANOVA; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0066] Figure 3 The effect of adding reagent CE during serum-free culture to maintain the growth phase on γδT cells; (A) γδT cell expansion fold; (B) γδT cell Vδ2 + (C) Proportion (purity, %); (D) γδT cell viability; (F) γδT cell intracellular ROS level (DCFH-DA mean fluorescence intensity MFI); Data are expressed as mean ± SD (n=3); Statistical analysis was performed using t-tests or one-way ANOVA; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0067] Figure 4 The effects of different recombinant human albumin (rAlb) concentrations on γδT cells; (A) γδT cell expansion fold; (B) γδT cell viability; (C) IFN-γ secretion (pg / mL); data are expressed as mean ± SD (n=3); statistical analysis was performed using one-way ANOVA; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0068] Figure 5 Comparison of different culture systems for expanding γδT cells; (A) γδT cells Vδ2 + (A) Proportion (purity, %); (B) IFN-γ secretion; (C) TNF-α secretion; (D) Kill rate at different effective-to-target ratios (E:T=10:1, 5:1, 1:1); Data are expressed as Mean ± SD (n=3); Statistical analysis was performed using one-way ANOVA; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0069] Figure 6 For morphological comparison of γδT cells under different culture systems.

[0070] Figure 7To eliminate the influence of different key components on γδT cell expansion and function; (A) γδT cell expansion fold; (B) IFN-γ secretion; (C) comparison of E:T=1:1 tumor killing rate; data are expressed as Mean ± SD (n=3); statistical analysis was performed using one-way ANOVA; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. Detailed Implementation

[0071] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention. Those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and these should also fall within the scope of protection claimed by the present invention. The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0072] Basic Example 1: Experimental Preparation and Procedure 1. Preparation of raw materials and solutions 1.1 Key Components of Serum-Free Culture Additives (1) Ethanolamine (cell culture grade): Prepare 100 mM aqueous solution and store at 4 ℃ protected from light; (2) Sodium selenite: Prepare 1 mM aqueous solution and store at 4 ℃ protected from light; (3) Recombinant human albumin: Cell culture grade, purchased from Wuhan Heyuan Biotechnology, catalog number HYC002M01, dissolved according to the instructions and filtered at 0.22 μm; (4) IL-15 (recombinant human): purchased from PeproTech, catalog number AF-200-15-1MG, resuspended in buffer according to the manufacturer's instructions, aliquoted and stored at -80℃; (5) IL-2: purchased from Beijing Sihuan Biotechnology, catalog number S20040008, resuspended in buffer according to the manufacturer's instructions, aliquoted and stored at -80℃.

[0073] 1.2 Serum-free culture additives (optional excipients) Optional excipients: L-alanyl-L-glutamine (200 mM), N-acetylcysteine ​​(1 M, pH 7.0), ascorbic acid (100 mM, freshly prepared), recombinant human transferrin (purchased from Wuhan Heyuan Biotechnology, catalog number HYC044M01, 10 mg / mL), recombinant human insulin (purchased from Beyotime, catalog number P3378-100 mg, 10 mg / mL).

[0074] 1.3 Basic Culture Medium The basal medium is RPMI-1640; if necessary, it can be mixed with IMDM at a volume ratio of 2:1 to enhance the buffer.

[0075] 2. Preparation of serum-free culture additives and culture medium 2.1 Serum-free culture additive stock solution Prepare “20× stock solutions” using basal culture medium at the following concentrations: ethanolamine stock solution: 4-24 μM; selenite stock solution: 0.8-3.2 μM; recombinant human albumin stock solution: 40-200 mg / mL; IL-15 stock solution: 40-200 ng / mL; IL-2 stock solution: 1000-3000 IU / mL; optional excipient stock solutions: prepared at working concentration × 20.

[0076] 2.2 Complete Culture Medium Basic culture medium: serum-free culture supplement = 19:1 v / v. After mixing, measure pH and osmotic pressure. pH: 7.4±0.1, osmotic pressure: 320±10 mOsm / kg; adjust with sterile NaCl or sterile water if necessary.

[0077] 3. Cell source and preparation Donor: Peripheral blood from healthy volunteers or patients; PBMC isolation: Ficoll gradient centrifugation; Cell seeding density: 0.8-1.5 × 10⁻⁶ 6 cells / mL; Cell equipment: disposable cell culture flasks.

[0078] Example 1: Effect of the molar ratio of ethanolamine to sodium selenite on γδT cells 1. Preparation of reagents for serum-free culture 1.1 Serum-free culture with the addition of reagent A and its culture medium The components of reagent A added for serum-free culture are shown in Table 1: Table 1

[0079] The above components were mixed with RPMI-1640 basal medium as solvent, and the pH was adjusted to 7.2-7.6. The mixture was then sterilized by 0.22 μm filtration to obtain serum-free culture additive A. Serum-free culture additive A in Example 1 contains: ethanolamine 4 μM, sodium selenite 0.8 μM, recombinant human albumin 100 mg / mL, IL-15 160 ng / mL, L-alanyl-L-glutamine 80 mM, N-acetylcysteine ​​20 mM, ascorbic acid 4 mM, recombinant human transferrin 100 mg / L, and recombinant human insulin 100 mg / L.

[0080] Serum-free culture medium A was prepared by mixing serum-free culture additive A with RPMI-1640 basal medium at a volume ratio of 1:19. The serum-free culture medium A had a pH of 7.4 and an osmotic pressure of 320 ± 10 mOsm / kg, and was sterilized by 0.22 μm filtration. The serum-free culture medium A in Example 1 contained: ethanolamine 200 nM, sodium selenite 40 nM, recombinant human albumin 5 mg / mL, IL-15 8 ng / mL, L-alanyl-L-glutamine 4 mM, N-acetylcysteine ​​1 mM, ascorbic acid 200 mM, recombinant human transferrin 5 mg / L, and recombinant human insulin 5 mg / L.

[0081] 1.2 Serum-free culture with the addition of reagent C and its culture medium The components of reagent C added for serum-free culture are shown in Table 2: Table 2

[0082] The above components were mixed using RPMI-1640 basal medium as solvent. The pH was adjusted to 7.2-7.6, and the mixture was sterilized by 0.22 μm filtration to obtain serum-free culture additive C. Serum-free culture additive C in Example 1 contains: ethanolamine 4 μM, sodium selenite 0.8 μM, recombinant human albumin 100 mg / mL, IL-15 160 ng / mL, IL-2 8000 IU / mL, L-alanyl-L-glutamine 80 mM, N-acetylcysteine ​​20 mM, ascorbic acid 4 mM, recombinant human transferrin 100 mg / L, and recombinant human insulin 100 mg / L.

[0083] Serum-free culture medium supplement C was mixed with RPMI-1640 basal medium at a volume ratio of 1:19 to obtain serum-free culture medium C. The pH of serum-free culture medium C was 7.4, and the osmotic pressure was 320 ± 10 mOsm / kg. It was sterilized by 0.22 μm filtration. The serum-free culture medium C of Example 1 contained: ethanolamine 200 nM, sodium selenite 40 nM, recombinant human albumin 5 mg / mL, IL-15 8 ng / mL, IL-2 400 IU / mL, L-alanyl-L-glutamine 4 mM, N-acetylcysteine ​​1 mM, ascorbic acid 200 mM, recombinant human transferrin 5 mg / L, and recombinant human insulin 5 mg / L.

[0084] 1.3 Serum-free culture with the addition of reagent D and its culture medium The components of reagent D added to serum-free culture are shown in Table 3: Table 3

[0085] The only difference between serum-free culture reagent D and serum-free culture reagent C is the concentration of IL-2. Similarly, the only difference between serum-free culture medium D and serum-free culture medium C is the concentration of IL-2. The preparation methods are the same as for serum-free culture reagent C and serum-free culture medium C.

[0086] 1.4 Serum-free culture with the addition of reagent E and its culture medium The components of reagent E added for serum-free culture are shown in Table 4: Table 4

[0087] The only difference between serum-free culture reagent E and serum-free culture reagent C is the concentration of IL-2. Similarly, the only difference between serum-free culture medium E and serum-free culture medium C is the concentration of IL-2. The preparation methods are the same as for serum-free culture reagent C and serum-free culture medium C.

[0088] 2. Verification process of the effect of the molar ratio of ethanolamine to sodium selenite on γδT cells 2.1 Cell source and inoculation PBMCs were isolated from peripheral blood of healthy donors, counted, and tested for viability ≥95% at a concentration of 1.0 × 10⁻⁶. 6 Inoculated at a density of cells / mL in serum-free culture medium A.

[0089] 2.2 Expansion of γδT cells (1) Activation phase (D0) Add 10 μM zoledronic acid and 400 IU / mL IL-2 to serum-free supplemented medium A that has been inoculated with PBMCs, and incubate at 37℃ and 5% CO2 for 24 h to activate the medium and obtain the activated medium.

[0090] (2) Segmented pulse supplementation (D1-D3) Serum-free culture additive B contains ethanolamine and sodium selenite, and the solvent is RPMI-1640 basal medium. Different molar ratios of serum-free culture additive B were prepared according to the molar ratios in Table 5. Table 5

[0091] Reagent B was added to the serum-free culture medium of groups 1-9 in three separate additions. The total target amount added in the three additions is shown in Table 5. Each addition was 1 / 3 of the total target amount added in the three additions, and the addition was done once every 24 hours.

[0092] (3) Maintenance growth stage (D4-D12) Cell density >2×10 6 When the cell count reaches 1 / mL, change the culture medium as follows: D4: Discard the original culture medium and replace it with serum-free culture medium C, maintaining a cell density of 1×10⁶ cells / year. 6 cells / mL; D7: Discard the original culture medium and replace it with serum-free supplemented medium D, maintaining a cell density of 1×10⁶ cells / mL; 6 cells / mL; D10: Discard the original culture medium and replace it with serum-free supplemented medium E, maintaining a cell density of 1×10⁶ cells / mL; 6 cells / mL.

[0093] (4) Harvest stage (D12–D14) Harvest cells and wash them; use them for in vitro functional testing or for downstream formulation steps.

[0094] 3. Quality Inspection On day 14, cell counts were used to determine the number of expanded cells, which were then converted to a fold increase from the initial cell count. Cell viability was calculated using AO / PI, and Vδ2 was measured using flow cytometry. + Proportion and survival rate. The test results are shown in Table 6. Statistical analysis was performed based on the results in Table 6, and the results are as follows: Figure 1 As shown.

[0095] Vδ2 expression purity as Figure 1 As shown in Figure A, 14 days after γδT cell expansion, there was no difference between groups 5, 6, 8, and 9 and group 1, indicating that when the molar ratio of ethanolamine to sodium selenite was between 3.75 and 12:1, there was no significant difference in Vδ2 cell purity after expansion, and the Vδ2 expression purity was >92% in all groups. Further multiple comparison analysis was performed among these five groups (…). Figure 1 In the B group, the Vδ2 expression purity of groups 1, 5, 8 and 9 is all >95%. Therefore, the preferred molar ratio of sodium selenite to ethanolamine is between 3.75 and 7.5.

[0096] Analysis of the fold expansion of γδT cells 14 days after expansion is shown in the figure. Figure 1 The results showed no significant differences among groups 1, 5, 6, 8, and 9, indicating that a molar ratio of ethanolamine to sodium selenite between 3.75 and 12:1 was effective in increasing cell expansion. Further multiple comparisons were performed among these five groups (…). Figure 1 (D) There was no difference in amplification fold among the groups.

[0097] Cell viability was detected in 9 groups of cells using AO / PI dyes, and the results are as follows: Figure 1 As shown in E, the overall viability of γδT cells in all nine groups remained at a high level (93.8%-97%), indicating that different ratios of ethanolamine to selenite had little overall impact on cell survival. Among them, the viability of groups with ratios between 3.75:1 and 12:1 (groups 1, 5, 6, 8, and 9) was all above 96%, and there was no significant difference between the groups (p>0.05).

[0098] The results indicate that within the optimal window range, the molar ratio of ethanolamine to selenite can stably maintain cell membrane integrity and metabolic homeostasis without causing significant cell damage. When the ratio is too high or too low, the cell viability in group 2 (15:1, group 3 (30:1), and group 7 (1.25:1) decreases slightly, with the most significant decreases in groups 3 and 7 (significantly lower than the optimal group, p<0.001 and p<0.01, respectively). This suggests that excessive amounts of ethanolamine or selenite can disrupt the redox balance of cells, leading to a slight decrease in cell viability.

[0099] In summary, the γδT cells cultured with a molar ratio of ethanolamine to selenite between 3.75 and 12:1 exhibit superior fold expansion, cell purity, and viability, with the preferred molar ratio between 3.75 and 7.5:1.

[0100] Table 6

[0101] Example 2: Comparison of staged and single-stage addition of ethanolamine and sodium selenite 1. Cell source and inoculation PBMCs were isolated from peripheral blood of healthy donors, counted, and tested for viability ≥95% at a concentration of 1.0 × 10⁻⁶. 6 The cells / mL were inoculated into the serum-free culture medium A obtained in Example 1.

[0102] 2. Expansion of γδT cells (1) Activation phase (D0): Add 10 μM zoledronic acid and 400 IU / mL IL-2 to serum-free supplemented medium A that has been inoculated with PBMCs, and incubate at 37℃ and 5% CO2 for 24 h to activate the medium and obtain the activated medium.

[0103] (2) Supplementation stage (D1-D3) Serum-free culture was supplemented with reagent B containing ethanolamine and sodium selenite, with a molar ratio of ethanolamine to sodium selenite of 6:1, and the solvent was the basal medium RPMI-1640.

[0104] The above serum-free culture reagent B was added to the activation medium in different ways, with a total target amount of 600 nM ethanolamine and 100 nM sodium selenite.

[0105] Group A: Serum-free culture was supplemented with reagent B, which was added to the activation medium in 3 portions. The total target amount for the 3 additions was 600 nM ethanolamine and 100 nM sodium selenite. Each addition was 1 / 3 of the total target amount for the 3 additions, and the addition was done once every 24 hours.

[0106] Group B: Serum-free culture with added reagent B, added to the activation medium on day D1, with a total target amount of 600 nM ethanolamine and 100 nM sodium selenite.

[0107] Group C: Serum-free culture was supplemented with reagent B, which was added to the activation medium in two portions. The total target amount for the two additions was 600 nM ethanolamine and 100 nM sodium selenite. 70% of the total target amount was added on D1 and 30% of the total target amount was added on D3.

[0108] (3) Refer to “(3) Maintenance growth stage (D4-D12)” in Example 1.

[0109] (4) Harvest stage (D12–D14) Harvest cells and wash them; use them for in vitro functional testing or for downstream formulation steps.

[0110] 3. Quality Inspection On day 14, cell counts were used to determine the number of expanded cells, which were then converted to a fold increase from the initial cell count. Cell viability was calculated using AO / PI, and Vδ2 was measured using flow cytometry. + Proportion, survival rate.

[0111] The test results are shown in Table 7 and Figure 2 As shown, the fold increase in γδT cells and Vδ2 in group A + The purity of group A was significantly higher than that of group B (p < 0.01), while there was no significant difference compared to group C (p > 0.05). Specifically, the average amplification fold of group A was 10¹⁰ ± 75-fold, and Vδ²... + The purity was 96.5 ± 1.0%. Group B had an amplification fold of only 730 ± 62 times and a purity of 91.0 ± 1.8%, while Group C was in between, with an amplification fold of 880 ± 68 times and a purity of 93.5 ± 1.2%. Cell viability remained between 95% and 97% in all three groups, with no significant difference (p > 0.05). These results indicate that the segmented pulsed supplementation of ethanolamine and sodium selenite can maintain antioxidant balance in the early stages of γδT cell activation, avoiding the oxidative stress peak caused by transient high concentrations, thereby significantly promoting cell expansion and Vδ2 subset purification without affecting cell viability.

[0112] Table 7

[0113] Example 3: Effect of serum-free culture with added reagent CE on γδT cells during the maintenance growth phase 1. Cell source and inoculation PBMCs were isolated from peripheral blood of healthy donors, counted, and tested for viability ≥95% at a concentration of 1.0 × 10⁻⁶. 6 The cells / mL were inoculated into the serum-free culture medium A obtained in Example 1.

[0114] 2. Expansion of γδT cells (1) Activation phase (D0): Add 10 μM zoledronic acid and 400 IU / mL IL-2 to serum-free supplemented medium A that has been inoculated with PBMCs, and incubate at 37℃ and 5% CO2 for 24 h to activate the medium and obtain the activated medium.

[0115] (2) Supplementation stage (D1-D3) Serum-free culture additive B contains ethanolamine and sodium selenite, with a molar ratio of ethanolamine to sodium selenite of 6:1. The solvent is RPMI-1640 basal medium. Serum-free culture additive B is added to the activation medium in three divided doses. The total target amount for the three additions is 600 nM ethanolamine and 100 nM sodium selenite. Each addition is one-third of the total target amount for the three additions, and the addition is performed every 24 hours.

[0116] (3) Maintenance growth stage (D4-D12) Cell density >2×10 6 When the cells / mL level is reached, different replacement strategies are used to replace the culture medium: ① Low-dose IL-2 tapering + IL-15 fixation (Method 1) D4: Discard the original culture medium and replace it with serum-free culture medium C obtained in Example 1, maintaining a cell density of 1×10⁶ cells / year. 6 cells / mL; D7: Discard the original culture medium and replace it with serum-free culture medium D obtained in Example 1, maintaining a cell density of 1×10⁶ cells / mL; 6 cells / mL; D10: Discard the original culture medium and replace it with serum-free culture medium E obtained in Example 1, maintaining a cell density of 1×10⁶ cells / mL; 6 cells / mL.

[0117] ② High-dose IL-2 + IL-15 fixation method (Method 2) D4: Discard the original culture medium and replace it with serum-free culture medium F, maintaining a cell density of 1×10⁶ cells / year. 6 cells / mL; D7: Discard the original culture medium and replace it with fresh serum-free supplemented culture medium F, maintaining a cell density of 1×10⁶ cells / mL; 6 cells / mL; D10: Discard the original culture medium and replace it with fresh serum-free supplemented culture medium F, maintaining a cell density of 1×10⁶ .... 6 cells / mL.

[0118] The components of serum-free culture additive F and serum-free culture medium F are shown in Table 8. The preparation methods are the same as those of serum-free culture additive C and serum-free culture medium C, with the only difference being the concentration of IL-2 added.

[0119] Table 8

[0120] (4) Harvest stage (D12–D14) Harvest cells and wash them; use them for in vitro functional testing or for downstream formulation steps.

[0121] 3. Quality Inspection After culture, the cell expansion fold and Vδ2 were measured. + Purity, cell viability, and intracellular ROS levels were measured. ROS was detected using the DCFH-DA fluorescent probe method: 1 × 10⁶ cells were used. 6 Cells were incubated with 10 μM DCFH-DA at 37°C in the dark for 30 min, washed with PBS, and then the mean fluorescence intensity (MFI) was measured by flow cytometry to reflect the level of oxidative stress. Cell morphology changes were observed under a microscope. All data are expressed as mean ± SD (n=3), and significance was compared using one-way ANOVA and Tukey's test. The results are shown in Table 9. Figure 3 As shown, the low-dose IL-2 decrementing + IL-15 fixation method (Method 1) significantly improved the expansion efficiency and purity of γδT cells compared to Method 2, while significantly reducing cellular oxidative stress levels. Regarding the expansion fold... Figure 3 In group A), the value of method 1 was 1080 ± 70 times, which was significantly higher than that of method 2 (760 ± 85 times) (p < 0.01); Vδ2 + purity( Figure 3 In group B, the rate reached 96.9 ± 0.8%, significantly higher than that in group 2 (89.5 ± 1.6%) (p < 0.01). ROS detection results ( Figure 3As shown in Figure C), the average fluorescence intensity of group 1 was 182 ± 14, which was significantly lower than that of group 2 (230 ± 18) by about 20% (p < 0.05), indicating that this strategy can effectively inhibit intracellular ROS accumulation. In summary, the combination of low-dose IL-2 decrementing and IL-15 fixation can significantly improve γδT cell purity, reduce ROS levels, and improve cell state while maintaining expansion efficiency.

[0122] Table 9

[0123] Example 4: Effect of recombinant human albumin concentration on γδT cells 1. Preparation of reagents for serum-free culture 1.1 Serum-free culture with the addition of reagents A1-A4 and their culture medium Serum-free culture supplements A1-A4 and serum-free culture media A1-A4 were prepared using the same methods as serum-free culture supplement A and serum-free culture media A in Example 1, differing only in the concentration of recombinant human albumin. The concentrations of recombinant human albumin in serum-free culture supplements A1-A4 and serum-free culture media A1-A4 are shown in Table 10. Table 10

[0124] 1.2 Serum-free culture with reagent B Serum-free culture was supplemented with reagent B containing ethanolamine and sodium selenite, with a molar ratio of ethanolamine to sodium selenite of 6:1, and the solvent was the basal medium RPMI-1640.

[0125] 1.3 Serum-free culture with the addition of reagents C1-C4 and their culture medium Serum-free culture supplements C1-C4 and serum-free culture media C1-C4 were prepared using the same methods as serum-free culture supplement C and serum-free culture media C in Example 1, differing only in the concentration of recombinant human albumin. The concentrations of recombinant human albumin in serum-free culture supplements C1-C4 and serum-free culture media C1-C4 are shown in Table 11. Table 11

[0126] 1.4 Serum-free culture with the addition of reagents D1-D4 and their culture medium Serum-free culture supplements D1-D4 and serum-free culture media D1-D4 were prepared using the same methods as serum-free culture supplement D and serum-free culture media D in Example 1, differing only in the concentration of recombinant human albumin. The concentrations of recombinant human albumin in serum-free culture supplements D1-D4 and serum-free culture media D1-D4 are shown in Table 12. Table 12

[0127] 1.5 Serum-free culture with the addition of reagents E1-E4 and their culture medium Serum-free culture was supplemented with reagents E1-E4, and the composition and preparation method of serum-free culture media E1-E4 were the same as those in Example 1, except for the concentration of recombinant human albumin. The concentrations of recombinant human albumin in serum-free culture media E1-E4 are shown in Table 13. Table 13

[0128] 2. Verification process of the effect of recombinant human albumin concentration on γδT cells Four groups were set up with recombinant human albumin concentrations of 0, 2, 5, and 10 mg / mL to verify the effect of recombinant human albumin concentration on γδT cells. The serum-free culture medium used in each group is shown in Table 14. Table 14

[0129] 2.1 Cell source and inoculation PBMCs were isolated from peripheral blood of healthy donors, counted, and tested for viability ≥95% at a concentration of 1.0 × 10⁻⁶. 6 Cells / mL were inoculated into serum-free culture media A1-A4 at densities of 1,000 cells / mL.

[0130] 2.2 Expansion of γδT cells (1) Activation phase (D0) Zoledronic acid 10 μM and IL-2 400 IU / mL were added to serum-free supplemented medium A1-A4 containing PBMCs, and the medium was incubated at 37℃ and 5% CO2 for 24 h to activate the PBMCs, thus obtaining different activation mediums.

[0131] (2) Segmented pulse supplementation (D1-D3) Add reagent B to serum-free culture medium in three separate additions to different activation media. The total target amount for the three additions is 600 nM ethanolamine and 100 nM sodium selenite. Each addition is 1 / 3 of the total target amount for the three additions, and the addition is made every 24 hours.

[0132] (3) Maintenance growth stage (D4-D12) Cell density >2×10 6 When the cells / mL level is reached, different replacement strategies are used to replace the culture medium: D4: Discard the original culture medium and replace it with serum-free medium C1 to serum-free medium C4, maintaining a cell density of 1×10⁻⁶ cells / year. 6 cells / mL; D7: Discard the original culture medium and replace it with serum-free supplemented medium D1 to serum-free supplemented medium D4, maintaining a cell density of 1×10⁶ cells / mL. 6 cells / mL; D10: Discard the original culture medium and replace it with serum-free supplemented medium E1 to serum-free supplemented medium E4 respectively, maintaining the cell density at 1×10⁶ cells / mL. 6 cells / mL.

[0133] (4) Harvest stage (D12–D14) Harvest cells and wash them; use them for in vitro functional testing or for downstream formulation steps.

[0134] 3. Quality Inspection After culture, the amplification rate, viability, and IFN-γ secretion level were measured. IFN-γ secretion level was determined using an IFN-γ ELISA kit (purchased from Dakota, catalog number 1110003). The results are shown in Table 15 and... Figure 4 As shown, appropriate amounts of recombinant human albumin significantly promote the expansion and metabolic homeostasis of γδT cells. In the 0 mg / mL group, cell expansion was limited under albumin-free conditions, with a 3%-5% decrease in viability. When the recombinant albumin concentration was 5 mg / mL, all indicators were optimal, with an expansion fold of 1085 ± 70 times, a viability of 97.3 ± 0.6%, and an increase in IFN-γ secretion of approximately 16%. While the 10 mg / mL group still maintained a high viability, cell expansion decreased slightly due to a slight increase in osmotic pressure. Comprehensive analysis shows that recombinant human albumin not only provides osmotic pressure regulation and carrier functions in this system but also binds fatty acids, trace elements, and oxidative metabolic byproducts, maintaining extracellular environmental stability and reducing ROS accumulation. Therefore, the optimal concentration range for recombinant human albumin is 3-7 mg / mL, preferably 5 mg / mL. γδT cells cultured within this concentration range exhibit higher expansion efficiency, metabolic stability, and functional consistency, making them suitable for clinical-grade cell preparation and scale-up production.

[0135] Table 15

[0136] Example 5: The method of serum-free culture with added reagents and culture medium combinations, and in vitro expansion of γδT cells according to the present invention. 1. Serum-free culture reagents and culture medium combinations The components of serum-free culture additive A and its serum-free culture medium A are shown in Table 1.

[0137] Serum-free culture was supplemented with reagent B containing ethanolamine and sodium selenite, with a molar ratio of ethanolamine to sodium selenite of 6:1, and the solvent was the basal medium RPMI-1640.

[0138] The components of serum-free culture additive C and its serum-free culture medium C are shown in Table 2.

[0139] The components of serum-free culture additive D and its serum-free culture medium D are shown in Table 3.

[0140] The components of serum-free culture additive E and its serum-free culture medium E are shown in Table 4.

[0141] 2. Methods for in vitro expansion of γδT cells 2.1 Cell source and inoculation PBMCs were isolated from peripheral blood of healthy donors, counted, and tested for viability ≥95% at a concentration of 1.0 × 10⁻⁶. 6 Inoculated at a density of cells / mL in serum-free culture medium A.

[0142] 2.2 Expansion of γδT cells (1) Activation phase (D0) Add 10 μM zoledronic acid and 400 IU / mL IL-2 to serum-free supplemented medium A that has been inoculated with PBMCs, and incubate at 37℃ and 5% CO2 for 24 h to activate the medium and obtain the activated medium.

[0143] (2) Segmented pulse supplementation (D1-D3) For serum-free culture, reagent B was added to the activation medium in three portions. The total target amount for the three additions was 600 nM ethanolamine and 100 nM sodium selenite. Each addition was one-third of the total target amount for the three additions, and the addition was made every 24 hours.

[0144] (3) Maintenance growth stage (D4-D12) Cell density >2×10 6 When the cell count reaches 1 / mL, change the culture medium as follows: D4: Discard the original culture medium and replace it with serum-free culture medium C, maintaining a cell density of 1×10⁶ cells / year. 6 cells / mL; D7: Discard the original culture medium and replace it with serum-free supplemented medium D, maintaining a cell density of 1×10⁶ cells / mL; 6 cells / mL; D10: Discard the original culture medium and replace it with serum-free supplemented medium E, maintaining a cell density of 1×10⁶ cells / mL; 6 cells / mL.

[0145] (4) Harvesting stage (D12-D14): Harvest cells and wash them.

[0146] Example 6: Validation of γδT cell tumor-killing function To verify whether γδT cells expanded using the serum-free culture reagent and culture medium combination of this invention maintain good tumor-killing function and cytokine secretion capacity, and to evaluate the advantages of this system in maintaining immune function, a comparison was made with the traditional culture system containing 10% fetal bovine serum (FBS) (RPMI-1640 + 10% FBS). Using the serum-free culture reagent and culture medium combination and method provided in Example 5, γδT cells were expanded in vitro. Cells were harvested 14 days after expansion and subjected to the following tests: (1) Flow cytometry detection of Vδ2 in γδT cells + (2) The killing rate of MDA-MB-231 tumor cells was determined by LDH method (E:T ratio was 10:1, 5:1, 1:1); (3) The content of IFN-γ and TNF-α in the culture supernatant was detected by ELISA method (pg / mL); (4) Cell morphology and colony status were observed under a microscope.

[0147] The experimental results are shown in Table 16 and Figure 5 As shown, the serum-free culture reagent and culture medium provided by this invention (Group A) exhibit significant advantages over the traditional culture system containing 10% fetal bovine serum (Group B) in terms of γδT cell purity, cytokine secretion, and in vitro tumor-killing activity. The Vδ2 of γδT cells in Group A... + The purity was 96.8 ± 0.9%, significantly higher than that of group B (91.2 ± 1.5%) (p < 0.01). Figure 5 (A) indicates that the serum-free culture additive reagent and its culture medium of the present invention can more effectively induce and maintain the specific expansion of γδT cells.

[0148] ELISA results showed that the IFN-γ secretion level of γδT cells in group A was 1890 ± 130 pg / mL, significantly higher than that in group B (1430 ± 120 pg / mL) (p < 0.05). Figure 5(B in the text). This result indicates that serum-free culture with added reagents and culture medium can maintain the functional activation state of γδT cells, promote the secretion of Th1 cytokines, and thus enhance the immune effector function of cells.

[0149] The TNF-α level in group A γδT cells was 980 ± 85 pg / mL, significantly higher than that in group B (835 ± 80 pg / mL) (p < 0.05). Figure 5 (C) shows that the serum-free culture reagent and its culture medium of the present invention can enhance the secretion capacity of inflammatory cytokines of γδT cells and further enhance their anti-tumor effect.

[0150] LDH assay results showed that group A γδT cells exhibited stronger cytotoxic activity against MDA-MB-231 tumor cells. Under E:T ratios of 10:1, 5:1, and 1:1, the killing rates of group A were 79.5 ± 3.0%, 64.8 ± 2.5%, and 52.1 ± 2.4%, respectively, all significantly higher than those of group B (66.2 ± 3.2%, 53.1 ± 2.8%, and 41.6 ± 2.3%, respectively) (p < 0.01). Figure 5 (D in the text). The results showed a clear effector-to-target ratio dependence, and group A was superior to the control group under all E:T conditions, indicating that the serum-free cultured γδT cells with added reagents and culture medium have higher specific killing ability and functional stability.

[0151] Table 16

[0152] Note: Group A in the table is the serum-free culture additive reagent and its culture medium of the present invention; Group B is the traditional culture system containing 10% fetal bovine serum (FBS) (RPMI-1640 + 10% FBS).

[0153] In this invention, γδT cells cultured in serum-free culture with added reagents and culture medium exhibited uniform, compact spherical colonies with clear cell boundaries, relatively consistent clump size, stable suspension, and transparent, highly refractive cytoplasm. In contrast, group B cell clumps were significantly larger and unevenly distributed, with some clumps collapsing and showing increased cell fragmentation. This morphological difference indicates that the system of this invention significantly improves the cell uniformity and structural stability of γδT cells during in vitro expansion by optimizing osmotic pressure, supplementing the antioxidant balance of ethanolamine and selenite, and utilizing the nutrient buffering function of recombinant albumin. Figure 6 ).

[0154] Example 7: Effects of key component removal on γδT cell expansion and function To further verify the necessity and synergistic effect of each key functional component in the serum-free system of the present invention, ethanolamine, selenite, recombinant human albumin (rAlb), or IL-15 were removed from the serum-free culture additives and culture medium formulations of Example 5, and their effects on γδT cell expansion efficiency, viability, functional cytokine secretion, and tumor-killing activity were evaluated, thereby determining the biological role of each component in the system.

[0155] The only difference between Comparative Example A and Example 5 is that ethanolamine was removed from the serum-free culture medium AE.

[0156] The only difference between Comparative Example B and Example 5 is that selenite was removed from the serum-free culture medium AE.

[0157] The only difference between Comparative Example C and Example 5 is that recombinant human albumin was removed from the serum-free culture medium AE.

[0158] The only difference between Comparative Example D and Example 5 is that IL-15 was removed from the serum-free culture medium AE.

[0159] Following the method for in vitro expansion of γδT cells described in Example 5, serum-free culture reagents and culture media from Example 5, and serum-free culture reagents and culture media from Comparative Example AD, were used to expand γδT cells in vitro. After 14 days of culture, the following parameters were measured: γδT cell expansion fold, cell viability, IFN-γ secretion, and tumor killing rate at E:T=1:1 (LDH method). Three biological replicates were performed for each group, and results are expressed as Mean ± SD.

[0160] The experimental results are shown in Table 17 and Figure 7 As shown:

[0161] Ethanolamine deficiency (Comparative Example A) resulted in a decrease of approximately 25% in cell expansion and IFN-γ secretion, and a 6% decrease in tumor killing rate. Ethanolamine is a precursor of cell membrane phospholipids; its deficiency restricts membrane synthesis and reduces cell proliferation and signal transduction capabilities, indicating that it is an essential component for maintaining cell viability and proliferation.

[0162] Selenite deficiency (Comparative Example B) led to a significant increase in early-stage (D3) apoptosis rate (p < 0.05), a decrease in cell proliferation rate by approximately 30%, and a significant reduction in IFN-γ secretion. Selenite is a cofactor of glutathione peroxidase; its deficiency disrupts cellular antioxidant balance, leading to ROS accumulation and metabolic disorders. It is a key factor in maintaining γδT cell survival and oxidative homeostasis.

[0163] The absence of recombinant human albumin (comparative example C) caused a 4% decrease in cell viability, a 25% increase in lactate accumulation, and uneven cell morphology. This indicates that recombinant human albumin not only provides osmotic pressure regulation and nutritional support, but also buffers system fluctuations by binding lipids, metal ions, and metabolic intermediates, making it a core component for maintaining metabolic stability and cell homogeneity.

[0164] IL-15 deficiency (Comparative Example D) leads to Vδ2 + The purity decreased by approximately 8%, IFN-γ secretion decreased by 18%, and the tumor killing rate at an E:T ratio of 1:1 decreased by 10%. This indicates that under low IL-2 conditions, IL-15 synergistically activates STAT5 signaling through the γ-chain receptor pathway, playing an important role in maintaining γδT cell function and memory-like phenotype.

[0165] The above results indicate that ethanolamine, selenite, recombinant human albumin, and IL-15 constitute the core synergistic module of the system of this invention, playing complementary roles in cell membrane synthesis, antioxidant balance, metabolic stability, and immune activation, respectively. Removal of any component leads to a decrease in amplification efficiency, functional cytokine secretion, and tumor-killing activity, demonstrating that the synergistic design of this system is irreplaceable.

[0166] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A defined serum-free supplement medium combination for γδ T cells, characterized in that, The serum-free additive culture medium combination comprises serum-free additive culture medium A, serum-free culture additive reagent B, serum-free additive culture medium C, serum-free additive culture medium D and serum-free additive culture medium E. The serum-free additive culture medium A is composed of ethanolamine, sodium selenite, recombinant human albumin, IL-15, an excipient combination and a basic culture medium. The serum-free culture additive reagent B is composed of ethanolamine and sodium selenite, wherein the molar ratio of ethanolamine to sodium selenite is 3.75-12:

1. The serum-free additive culture medium C, serum-free additive culture medium D and serum-free additive culture medium E are composed of ethanolamine, sodium selenite, recombinant human albumin, IL-15, IL-2, an excipient combination and a basic culture medium; the content of IL-2 in the serum-free culture additive C is 350-450 IU / mL; the content of IL-2 in the serum-free culture additive D is 150-250 IU / mL; and the content of IL-2 in the serum-free additive culture medium E is 50-150 IU / mL.

2. The serum-additive-free medium combination according to claim 1, characterized in that, The serum-free culture additive reagent B is added in an equal molar amount in three times, and the total target amount is 200-1200 nM of ethanolamine and 40-160 nM of sodium selenite.

3. The serum-additive-free medium combination according to claim 1, characterized in that, The serum-free culture additive reagent A contains 100-300 nM of ethanolamine, 20-60 nM of sodium selenite, 2.5-7.5 mg / mL of recombinant human albumin and 6-10 ng / mL of IL-15; or the serum-free culture additive reagent C contains 100-300 nM of ethanolamine, 20-60 nM of sodium selenite, 2.5-7.5 mg / mL of recombinant human albumin, 6-10 ng / mL of IL-15 and 350-450 IU / mL of IL-2; or the serum-free additive culture medium D contains 100-300 nM of ethanolamine, 20-60 nM of sodium selenite, 2.5-7.5 mg / mL of recombinant human albumin, 6-10 ng / mL of IL-15 and 150-250 IU / mL of IL-2; or the serum-free additive culture medium E contains 100-300 nM of ethanolamine, 20-60 nM of sodium selenite, 2.5-7.5 mg / mL of recombinant human albumin, 6-10 ng / mL of IL-15 and 50-150 IU / mL of IL-2.

4. The serum-additive-free medium combination according to claim 1, characterized in that, The excipient combination comprises L-alanyl-L-glutamine, N-acetylcysteine, ascorbic acid, recombinant human transferrin and recombinant human insulin.

5. The serum-additive free media combination of claim 1, wherein, The basic culture medium comprises any one or more of RPMI-1640 medium, IMDM medium, DMEM / F12 medium and α-MEM medium.

6. Use of the serum-free additive culture medium combination of any one of claims 1-5 in the preparation of a product of expanded γδ T cells in vitro.

7. A product for expanding γδ T cells in vitro, characterized in that, The product comprises the serum-free additive culture medium combination of any one of claims 1-5.

8. A method of expanding gd T cells in vitro, characterized in that, The method comprises culturing with the serum-free supplement medium combination according to any one of claims 1-5 or the product according to claim 7.

9. The method of claim 8, wherein, The gamma delta T cells prepared by the method are used for any one or more of preventing and / or treating tumor immunity, preventing and / or treating infectious diseases, and immunomodulation.

10. The gamma delta T cells prepared by the method according to any one of claims 8-9.

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