A method and culture medium for γδ T cells

Abstract

The application provides a kind of gamma delta T cell culture method and culture medium, belong to T cell culture technical field.In 1640 culture solution is added modified nanoparticle, zoledronic acid, vitamin C, glutamine, wherein, the concentration of the modified nanoparticle is 0.5-1g / L, the concentration of the zoledronic acid is 0.5-1.5 μmol / L, the concentration of the vitamin C is 200-500 μmol / L, the concentration of the glutamine is 0.1-0.2 mmol / L.The culture medium prepared by the application can efficiently stimulate gamma delta T cell expansion and improve its killing activity, and the survival rate of gamma delta T cells is also improved, and the culture method of gamma delta T cells is simple, the cost is low, and has broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of T cell culture technology, specifically to a method and culture medium for culturing γδ T cells. Background Technology

[0002] In human peripheral blood, T lymphocytes account for 65%-75% of the total number of lymphocytes and play a crucial role in the body's immune defense. T lymphocytes are divided into TCR αβ T cells (αβ T cells) and TCR γδ T cells (γδ T cells). γδ T cells are the T cells that perform innate immune functions. Their TCR (T Cell Receptor) is composed of γ and δ chains. γδ T cells can directly recognize and bind antigen molecules without major histocompatibility complex (MHC) restriction, and kill tumor cells and virus-infected cells through pathways such as IFN-γ secretion, perforin-granzyme, TNF-related apoptosis-inducing ligand receptors, and Fas / FasL. Cytokines such as IFN-γ secreted by γδ T cells can activate other immune cells, thus playing a vital immune role. γδ T cells express TCRγδ and NKG2D receptors on their surface, and it is these two receptors that exert the killing effect on tumor cells. γδ T cells are rapidly activated by TCRγδ and NKG2D when the body defends against tumors or infections. Activated γδ T cells then exert their anti-tumor or anti-infective effects. γδ T cells show significant therapeutic effects against mucosal cancers, particularly those of the respiratory, digestive, and reproductive systems. Because γδ T cells are not MHC-restricted in killing tumor cells, they exhibit significant cytotoxic activity against various autologous and allogeneic tumor cells, making them a highly anticipated candidate cell line for adoptive immunotherapy in tumor treatment.

[0003] Since γδ T cells account for approximately 0.5-5% of the total number of peripheral blood T lymphocytes in adults, obtaining large quantities of highly pure and active γδ T cells is extremely difficult. Conventional methods for expanding αβ T cells, such as anti-CD3 antibodies and IL-2, typically fail to achieve specific expansion of γδ T cells. Currently, in vitro expansion methods for γδ T cells can be mainly divided into two approaches: those based on the K562 feeder cell line and those based on biological bisphosphonates. However, the feeder cell line-based expansion method requires the introduction of tumor cells, posing potential safety risks and requiring strict quality control, thus raising concerns for its clinical application. Summary of the Invention

[0004] The purpose of this invention is to propose a γδ T cell culture method and culture medium that can efficiently stimulate the expansion of γδ T cells and improve their cytotoxic activity. At the same time, the survival rate of γδ T cells is also improved. Furthermore, the γδ T cell culture method is simple, low in cost, and has broad application prospects.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a culture medium in which modified nanoparticles, zoledronic acid, vitamin C, and glutamine are added to a 1640 culture medium. The concentration of the modified nanoparticles is 0.5-1 g / L, the concentration of the zoledronic acid is 0.5-1.5 μmol / L, the concentration of the vitamin C is 200-500 μmol / L, and the concentration of the glutamine is 0.1-0.2 mmol / L.

[0007] As a further improvement of the present invention, the method for preparing the modified nanoparticles is as follows:

[0008] S1. Magnetic nanoparticles were added to Tris-HCl solution, dopamine hydrochloride was added, the mixture was heated and stirred to react, separated by magnets, washed, and dried to obtain modified magnetic nanoparticles.

[0009] S2. Modified magnetic nanoparticles, zirconium chloride and 1,2,4,5-benzenetetracarboxylic acid were added to water, heated and stirred under reflux, magnetically separated, activated, magnetically separated, washed and dried to obtain UiO-66-(COOH)2@magnetic nanoparticles;

[0010] S3. Add UiO-66-(COOH)2@magnetic nanoparticles to water, add NHS and EDC, stir to activate, add disodium pamidronate and alendronate, stir to react, separate with a magnet, wash, dry, and obtain phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles;

[0011] S4. Add the interleukin composition to water, add NHS and EDC, stir to activate, add phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles, stir to react, separate with a magnet, wash, dry, and obtain modified nanoparticles.

[0012] As a further improvement of the present invention, the pH value of the Tris-HCl solution in step S1 is 8.5-9.5, the magnetic nanoparticles are iron oxide nanoparticles with a particle size of 20-50 nm, the mass ratio of the magnetic nanoparticles to dopamine hydrochloride is 10:1-2, the temperature of the heating and stirring reaction is 50-60℃, and the time is 3-5 h.

[0013] As a further improvement of the present invention, the mass ratio of the modified magnetic nanoparticles, zirconium chloride and 1,2,4,5-benzenetetracarboxylic acid in step S2 is 10:2-2.5:4-4.7, the heating and reflux stirring treatment is carried out for 20-24 hours, and the activation method is to add water and heat and reflux for 10-15 hours.

[0014] As a further improvement of the present invention, the mass ratio of UiO-66-(COOH)2@magnetic nanoparticles, NHS, EDC, disodium pamidronate and alendronate in step S3 is 12-15:2-4:2-4:1-2:0.8-1.2.

[0015] As a further improvement of the present invention, the interleukin composition in step S4 includes IL-15, IL-2 and IL-7 in a mass ratio of 2-3:5-7:1-2, and the mass ratio of the interleukin composition, NHS, EDC and phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles is 1-2:0.5-1:0.5-1:10-15.

[0016] This invention further protects a method for culturing γδ T cells, comprising the following steps:

[0017] (1) Preparation of peripheral blood human mononuclear cells: peripheral blood was collected, heparin was used for anticoagulation, and human mononuclear cells were separated by density gradient centrifugation with lymphocyte separation solution. The cells were washed with PBS solution and centrifuged to obtain peripheral blood human mononuclear cells.

[0018] (2) Isolation and purification of γδ T cells: peripheral blood human mononuclear cells were added to the above culture medium and cultured for 1-2 days. Anti-human gamma-delta TCR-FITC antibody-labeled γδ T cells were added, centrifuged, and the above culture medium was added. γδ T cells were then isolated and purified by flow cytometry.

[0019] (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium above, the cell density was adjusted, and the cells were treated with low-frequency ultrasound superimposed with magnetic field. After culturing for 2-4 days, the cells were irradiated with near-infrared light for 20-40 minutes and centrifuged to obtain stimulated γδ T cells.

[0020] (4) Expansion of γδ T cells: Stimulated γδ T cells are added to the culture medium described above, the cell density is adjusted, and culture is continued. The medium is replenished every 2-3 days according to the cell density until 12-17 days of culture is obtained to obtain highly active γδ T cells.

[0021] As a further improvement of the present invention, the step (3) of adjusting the cell density to 10 5 -10 6The low-frequency ultrasonic superimposed magnetic field treatment is performed by superimposing a 10-30mT magnetic field with a 30-50W ultrasonic wave for 1-2 days, and the wavelength of the near-infrared light is 808nm.

[0022] As a further improvement of the present invention, the adjustment of cell density to 10 in step (4) is described. 6 -10 7 Cells / mL, after which the cell density is maintained at 10⁶ / mL. 6 -10 7 per mL.

[0023] As a further improvement of the present invention, the culture conditions are 36-38℃, CO2 concentration of 3-7%, oxygen concentration of 25-35%, and the balance is nitrogen, where % is volume percentage.

[0024] The present invention has the following beneficial effects:

[0025] Zoledronic acid (ZOL) is a biological bisphosphonate commonly used to treat osteoporosis. ZOL shares structural homology with the non-peptide ligands of γδ T cells. Adding ZOL to human peripheral blood mononuclear cell (PBMC) cultures leads to the accumulation of isopentenyl pyrophosphate (IPP) in the mononuclear cells, activating γδ T cells through cell-cell interactions. However, γδ T cells expanded using zoledronic acid in combination with IL-2 have a short survival time, surviving only about 7 days after expansion. These cells exhibit weak anti-apoptotic activity, limiting their persistence and efficacy in clinical applications. Furthermore, there are drawbacks such as long culture periods and weak tumor-killing ability of the expanded γδ T cells.

[0026] This invention modifies the surface of magnetic iron oxide nanoparticles with polydopamine, then deposits a layer of UiO-66-(COOH)2 in situ on the surface. This layer has infrared excitation efficiency and can emit mid-infrared wavelengths in the culture medium, efficiently converting infrared light into heat energy to generate a thermal radiation field that stimulates cells. Simultaneously, under the superimposed stimulation of magnetic and ultrasonic fields, changes in the microenvironment temperature can induce mechanical stimulation, promoting cell diffusion and nuclear translocation of transcription factors. In the presence of interleukins and zoledronic acid, it can synergistically activate γδ T cells, improving proliferation efficiency and cell survival rate. Moreover, the method is simple and safe.

[0027] In addition, the carboxyl groups on the surface of the prepared UiO-66-(COOH)2@magnetic nanoparticles can react with the amino groups of pamidronate disodium and alendronate, thereby coupling the two to the surface of the nanospheres. Pamidronate disodium and alendronate not only have a bisphosphonic acid structure similar to zoledronic acid, which can stimulate the accumulation of isopentenyl pyrophosphate (IPP) in monocytes and activate γδ T cells through intercellular interactions, but also have high biosafety and, under synergistic effect, can effectively improve the γδ T cell proliferation efficiency and enhance activity.

[0028] Interleukin-2 (IL-2), as an immune response agonist, is an important cytokine that promotes the activation and expansion of T cells and plays an indispensable role in different T cell expansion systems. However, high concentrations of IL-2 can cause severe cytotoxicity, which limits its clinical application.

[0029] This invention significantly reduces the amount of IL-2 required by adding a composition of interleukins, including IL-15, IL-2, and IL-7, thereby reducing cytotoxicity. IL-2 and IL-15 synergistically promote CD69 expression in γδT cells, significantly improving their expansion efficiency. IL-15 can also maintain a long-lasting and effective T cell response by preserving CD8+ T cell survival, further enhancing γδT cell expansion efficiency. However, IL-15's short half-life and low biological affinity limit its biotherapeutic applications. The addition of IL-7, a necessary factor for T cell differentiation, plays a crucial role in T cell development, proliferation, and homeostasis regulation. The synergistic effect of these three components enhances T cell viability and improves expansion efficiency.

[0030] This invention couples an interleukin composition to the surface of phosphonic acid-modified UiO-66-(COOH)2@magnetic nanoparticles. On the one hand, this reduces the concentration of IL-2 in the solution, thus reducing cytotoxicity. On the other hand, by complexing IL-15 and coupling it with the carboxyl groups on the surface of the nanospheres, the molecular weight is increased, the half-life is prolonged, and the thermal stability is improved. Therefore, it also compensates for the short half-life of IL-15. The nanospheres have good biocompatibility, thereby greatly improving their biological effects.

[0031] The modified nanoparticles prepared by this invention can be added to a culture medium. After cell culture is completed, the modified nanoparticles can be separated by a magnet. After activation or re-coupling with active substances, they can be reused, thereby greatly reducing costs and improving utilization.

[0032] The culture medium prepared by this invention can efficiently stimulate the expansion of γδT cells and improve their killing activity. At the same time, the survival rate of γδT cells is also improved. In addition, the culture method of γδT cells is simple and low in cost, and has broad application prospects. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] NHS stands for N-hydroxysuccinimide; EDC stands for 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The magnetic nanoparticles are iron oxide nanoparticles with a particle size of 20-50 nm.

[0035] Preparation Example 1: Modified Nanoparticles

[0036] The preparation method is as follows:

[0037] S1. Add 1g of magnetic nanoparticles to 50mL of Tris-HCl solution, add 0.1g of dopamine hydrochloride, heat to 50℃, stir and react for 3h, separate the magnets, wash, dry, and obtain modified magnetic nanoparticles.

[0038] S2. 1g of modified magnetic nanoparticles, 0.2g of zirconium chloride and 0.4g of 1,2,4,5-benzenetetracarboxylic acid were added to 50mL of water, heated and stirred under reflux for 20h, and the magnetic nanoparticles were separated. The nanoparticles were then added to 50mL of water and heated under reflux for 10h, and the magnetic nanoparticles were separated, washed and dried to obtain UiO-66-(COOH)2@magnetic nanoparticles.

[0039] S3. Add 1.2g of UiO-66-(COOH)2@magnetic nanoparticles to 100mL of water, add 0.2g of NHS and 0.2g of EDC, stir and activate for 20min, add 0.1g of disodium pamidronate and 0.08g of alendronate, stir and react for 12h, separate with a magnet, wash, dry, and obtain phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles;

[0040] S4. Add 0.1g of interleukin composition to 50mL of water, add 0.05g of NHS and 0.05g of EDC, stir and activate for 20min, add 1g of phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles, stir and react for 12h, separate with magnets, wash, dry, and obtain modified nanoparticles;

[0041] The interleukin composition comprises IL-15, IL-2 and IL-7 in a mass ratio of 2:5:1.

[0042] Preparation Example 2: Modified Nanoparticles

[0043] The preparation method is as follows:

[0044] S1. Add 1g of magnetic nanoparticles to 50mL of Tris-HCl solution, add 0.2g of dopamine hydrochloride, heat to 60℃, stir and react for 5h, separate the magnets, wash, dry, and obtain modified magnetic nanoparticles.

[0045] S2. 1g of modified magnetic nanoparticles, 0.25g of zirconium chloride and 0.47g of 1,2,4,5-benzenetetracarboxylic acid were added to 50mL of water, heated and stirred under reflux for 24h, and the magnetic nanoparticles were separated. The nanoparticles were then added to 50mL of water and heated under reflux for 15h, and the magnetic nanoparticles were separated, washed and dried to obtain UiO-66-(COOH)2@magnetic nanoparticles.

[0046] S3. Add 1.5g of UiO-66-(COOH)2@magnetic nanoparticles to 100mL of water, add 0.4g of NHS and 0.4g of EDC, stir and activate for 30min, add 0.2g of disodium pamidronate and 0.12g of alendronate, stir and react for 15h, separate with a magnet, wash, dry, and obtain phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles;

[0047] S4. Add 0.2g of interleukin composition to 50mL of water, add 0.1g of NHS and 0.1g of EDC, stir and activate for 30min, add 1.5g of phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles, stir and react for 15h, separate with magnets, wash, dry, and obtain modified nanoparticles;

[0048] The interleukin composition comprises IL-15, IL-2, and IL-7 in a mass ratio of 3:7:2.

[0049] Preparation Example 3: Modified Nanoparticles

[0050] The preparation method is as follows:

[0051] S1. Add 1g of magnetic nanoparticles to 50mL of Tris-HCl solution, add 0.15g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified magnetic nanoparticles.

[0052] S2. 1g of modified magnetic nanoparticles, 0.22g of zirconium chloride and 0.42g of 1,2,4,5-benzenetetracarboxylic acid were added to 50mL of water, heated and stirred under reflux for 22h, and the magnetic nanoparticles were separated. The nanoparticles were then added to 50mL of water and heated under reflux for 12h, and the magnetic nanoparticles were separated, washed and dried to obtain UiO-66-(COOH)2@magnetic nanoparticles.

[0053] S3. Add 1.35g of UiO-66-(COOH)2@magnetic nanoparticles to 100mL of water, add 0.3g of NHS and 0.3g of EDC, stir and activate for 25min, add 0.15g of disodium pamidronate and 0.1g of alendronate, stir and react for 13h, separate with a magnet, wash, dry, and obtain phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles;

[0054] S4. Add 0.15g of interleukin composition to 50mL of water, add 0.07g of NHS and 0.07g of EDC, stir and activate for 25min, add 1.2g of phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles, stir and react for 13h, separate with magnets, wash, dry, and obtain modified nanoparticles;

[0055] The interleukin composition comprises IL-15, IL-2, and IL-7 in a mass ratio of 2.5:6:1.5.

[0056] Comparative Preparation Example 1

[0057] The difference from preparation example 3 is that step S2 was not performed.

[0058] The preparation method is as follows:

[0059] S1. Add 1g of magnetic nanoparticles to 50mL of Tris-HCl solution, add 0.15g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified magnetic nanoparticles.

[0060] S2. Add 1.35g of modified magnetic nanoparticles to 100mL of water, add 0.15g of disodium pamidronate and 0.1g of alendronate, heat to 45℃, stir and react for 13h, separate with magnets, wash, dry, and obtain phosphonic acid modified magnetic nanoparticles.

[0061] S3. Add 0.15g of interleukin composition to 50mL of water, add 0.07g of NHS and 0.07g of EDC, stir and activate for 25min, add 1.2g of phosphonic acid modified magnetic nanoparticles, stir and react for 13h, separate with magnets, wash, dry, and obtain modified nanoparticles.

[0062] The interleukin composition comprises IL-15, IL-2, and IL-7 in a mass ratio of 2.5:6:1.5.

[0063] Comparative Preparation Example 2

[0064] The difference from preparation example 3 is that step S3 was not performed.

[0065] The preparation method is as follows:

[0066] S1. Add 1g of magnetic nanoparticles to 50mL of Tris-HCl solution, add 0.15g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified magnetic nanoparticles.

[0067] S2. 1g of modified magnetic nanoparticles, 0.22g of zirconium chloride and 0.42g of 1,2,4,5-benzenetetracarboxylic acid were added to 50mL of water, heated and stirred under reflux for 22h, and the magnetic nanoparticles were separated. The nanoparticles were then added to 50mL of water and heated under reflux for 12h, and the magnetic nanoparticles were separated, washed and dried to obtain UiO-66-(COOH)2@magnetic nanoparticles.

[0068] S3. Add 0.15g of interleukin composition to 50mL of water, add 0.07g of NHS and 0.07g of EDC, stir and activate for 25min, add 1.2g of UiO-66-(COOH)2@magnetic nanoparticles, stir and react for 13h, separate with magnets, wash, dry, and obtain modified nanoparticles;

[0069] The interleukin composition comprises IL-15, IL-2, and IL-7 in a mass ratio of 2.5:6:1.5.

[0070] Comparative preparation example 3

[0071] The difference compared to preparation example 3 is that step S4 was not performed.

[0072] The preparation method is as follows:

[0073] S1. Add 1g of magnetic nanoparticles to 50mL of Tris-HCl solution, add 0.15g of dopamine hydrochloride, heat to 55℃, stir and react for 4h, separate the magnets, wash, dry, and obtain modified magnetic nanoparticles.

[0074] S2. 1g of modified magnetic nanoparticles, 0.22g of zirconium chloride and 0.42g of 1,2,4,5-benzenetetracarboxylic acid were added to 50mL of water, heated and stirred under reflux for 22h, and the magnetic nanoparticles were separated. The nanoparticles were then added to 50mL of water and heated under reflux for 12h, and the magnetic nanoparticles were separated, washed and dried to obtain UiO-66-(COOH)2@magnetic nanoparticles.

[0075] S3. Add 1.35g of UiO-66-(COOH)2@ magnetic nanoparticles to 100mL of water, add 0.3g of NHS and 0.3g of EDC, stir and activate for 25min, add 0.15g of disodium pamidronate and 0.1g of alendronate, stir and react for 13h, separate with a magnet, wash, dry, and obtain phosphonic acid modified UiO-66-(COOH)2@ magnetic nanoparticles, which are the modified nanoparticles.

[0076] Example 1

[0077] This embodiment provides a culture medium in which modified nanoparticles prepared in Preparation Example 1, zoledronic acid, vitamin C, and glutamine are added to a 1640 culture medium. The concentration of the modified nanoparticles is 0.5 g / L, the concentration of the zoledronic acid is 0.5 μmol / L, the concentration of the vitamin C is 200 μmol / L, and the concentration of the glutamine is 0.1 mmol / L.

[0078] Example 2

[0079] This embodiment provides a culture medium in which modified nanoparticles prepared in Preparation Example 2, zoledronic acid, vitamin C, and glutamine are added to a 1640 culture medium. The concentration of the modified nanoparticles is 1 g / L, the concentration of the zoledronic acid is 1.5 μmol / L, the concentration of the vitamin C is 500 μmol / L, and the concentration of the glutamine is 0.2 mmol / L.

[0080] Example 3

[0081] This embodiment provides a culture medium in which modified nanoparticles prepared in Preparation Example 3, zoledronic acid, vitamin C, and glutamine are added to a 1640 culture medium. The concentration of the modified nanoparticles is 0.7 g / L, the concentration of the zoledronic acid is 1 μmol / L, the concentration of the vitamin C is 350 μmol / L, and the concentration of the glutamine is 0.15 mmol / L.

[0082] Comparative Example 1

[0083] The difference from Example 3 is that the modified nanoparticles were prepared by Comparative Preparation Example 1.

[0084] Comparative Example 2

[0085] The difference from Example 3 is that the modified nanoparticles were prepared by Comparative Preparation Example 2.

[0086] Comparative Example 3

[0087] The difference from Example 3 is that the modified nanoparticles were prepared by Comparative Preparation Example 3.

[0088] Example 4

[0089] This embodiment provides a method for culturing γδ T cells, including the following steps:

[0090] (1) Preparation of peripheral blood human mononuclear cells: peripheral blood was collected, heparin was used for anticoagulation, and human mononuclear cells were separated by density gradient centrifugation with lymphocyte separation solution. The cells were washed with PBS solution and centrifuged to obtain peripheral blood human mononuclear cells.

[0091] (2) Isolation and purification of γδ T cells: Peripheral blood human mononuclear cells were added to the culture medium prepared in Example 1 and cultured for 1 day. Anti-human gamma-delta TCR-FITC antibody-labeled γδ T cells were added, centrifuged, and the culture medium prepared in Example 1 was added. The γδ T cells were then isolated and purified by flow cytometry.

[0092] (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium prepared in Example 1, and the cell density was adjusted to 10. 5 Cells / mL were treated with 50W ultrasound superimposed with a 30mT magnetic field for 1 day, cultured for 4 days, and then irradiated with near-infrared light at a wavelength of 808nm for 40 minutes. After centrifugation, stimulated γδ T cells were obtained.

[0093] (4) Expansion of γδ T cells: Stimulated γδ T cells were added to the culture medium prepared in Example 1, and the cell density was adjusted to 10T. 6 Cells / mL, continue culturing, and replenish medium every 3 days according to cell density to maintain a cell density of 10. 6 Cells / mL were cultured until 17 days to obtain highly active γδ T cells;

[0094] The culture conditions were 36℃, CO2 concentration of 3%, oxygen concentration of 25%, and the remainder was nitrogen. Here, % refers to volume percentage.

[0095] Example 5

[0096] This embodiment provides a method for culturing γδ T cells, including the following steps:

[0097] (1) Preparation of peripheral blood human mononuclear cells: peripheral blood was collected, heparin was used for anticoagulation, and human mononuclear cells were separated by density gradient centrifugation with lymphocyte separation solution. The cells were washed with PBS solution and centrifuged to obtain peripheral blood human mononuclear cells.

[0098] (2) Isolation and purification of γδ T cells: Peripheral blood human mononuclear cells were added to the culture medium prepared in Example 2 and cultured for 2 days. Anti-human gamma-delta TCR-FITC antibody-labeled γδ T cells were added, centrifuged, and the culture medium prepared in Example 2 was added. The γδ T cells were then isolated and purified by flow cytometry.

[0099] (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium prepared in Example 2, and the cell density was adjusted to 10. 6Cells / mL were treated with 30W ultrasound superimposed with a 10mT magnetic field for 2 days, cultured for 2 days, and then irradiated with near-infrared light at a wavelength of 808nm for 20 minutes. After centrifugation, stimulated γδ T cells were obtained.

[0100] (4) Expansion of γδ T cells: Stimulated γδ T cells were added to the culture medium prepared in Example 2, and the cell density was adjusted to 102. 7 Cells / mL, continue culturing, and replenish medium every 2 days according to cell density to maintain a cell density of 10. 7 Cells / mL were cultured until 17 days to obtain highly active γδ T cells;

[0101] The culture conditions were 38℃, CO2 concentration of 7%, oxygen concentration of 35%, and the remainder was nitrogen. Here, % refers to volume percentage.

[0102] Example 6

[0103] This embodiment provides a method for culturing γδ T cells, including the following steps:

[0104] (1) Preparation of peripheral blood human mononuclear cells: peripheral blood was collected, heparin was used for anticoagulation, and human mononuclear cells were separated by density gradient centrifugation with lymphocyte separation solution. The cells were washed with PBS solution and centrifuged to obtain peripheral blood human mononuclear cells.

[0105] (2) Isolation and purification of γδ T cells: Peripheral blood human mononuclear cells were added to the culture medium prepared in Example 3 and cultured for 1.5 days. Anti-human gamma-delta TCR-FITC antibody-labeled γδ T cells were added, centrifuged, and the culture medium prepared in Example 3 was added. The γδ T cells were then isolated and purified by flow cytometry.

[0106] (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium prepared in Example 3, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5 The cells were treated with 40W ultrasound superimposed with a 20mT magnetic field for 1.5 days, cultured for 3 days, and then irradiated with near-infrared light at a wavelength of 808nm for 30 minutes. After centrifugation, stimulated γδ T cells were obtained.

[0107] (4) Expansion of γδ T cells: Stimulated γδ T cells were added to the culture medium prepared in Example 3, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 6 Cells / mL, continue culturing, and replenish medium every 2 days according to cell density to maintain a cell density of 5 × 10⁶ cells / mL. 6 Cells / mL were cultured until 17 days to obtain highly active γδ T cells;

[0108] The culture conditions were 37℃, CO2 concentration of 5%, oxygen concentration of 30%, and the remainder was nitrogen. Here, % refers to volume percentage.

[0109] Comparative Example 4

[0110] The difference from Example 6 is that the culture medium was prepared from Comparative Example 1.

[0111] Comparative Example 5

[0112] The difference from Example 6 is that the culture medium was prepared from Comparative Example 2.

[0113] Comparative Example 6

[0114] The difference from Example 6 is that the culture medium was prepared from Comparative Example 3.

[0115] Comparative Example 7

[0116] The difference from Example 6 is that 1640 culture medium was used instead of the culture medium.

[0117] Comparative Example 8

[0118] The difference from Example 6 is that near-infrared light irradiation was not performed in step (3).

[0119] Specifically as follows:

[0120] (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium prepared in Example 3, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5 Cells were cultured at a density of 1 cell / mL and treated with 40W ultrasound superimposed with a 20mT magnetic field for 1.5 days, followed by 3 days of culture and centrifugation to obtain stimulated γδ T cells.

[0121] Comparative Example 9

[0122] The difference from Example 6 is that no magnetic field treatment was performed in step (3).

[0123] Specifically as follows:

[0124] (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium prepared in Example 3, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5 Cells were collected at a density of 1 / mL, treated with 40W sonication for 1.5 days, cultured for 3 days, and then irradiated with near-infrared light at a wavelength of 808nm for 30 minutes. After centrifugation, stimulated γδ T cells were obtained.

[0125] Comparative Example 10

[0126] The difference from Example 6 is that no ultrasonic treatment was performed in step (3).

[0127] Specifically as follows:

[0128] (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium prepared in Example 3, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5 Cells were treated with a magnetic field of 20 mT for 1.5 days, cultured for 3 days, and then irradiated with near-infrared light at a wavelength of 808 nm for 30 minutes. After centrifugation, stimulated γδ T cells were obtained.

[0129] Test Example 1

[0130] The survival rate and in vitro expansion efficiency of highly active γδ T cells prepared by the methods in Examples 4-6 and Comparative Examples 4-10 were calculated. The results are shown in Table 1.

[0131] Amplification efficiency = (Total number of cells after culture × Proportion of γδ T cells to total number of cells) / Number of purified γδ T cells

[0132] Table 1

[0133]

[0134] As shown in the table above, the highly active γδ T cells prepared in Examples 4-6 of this invention have a high survival rate and high amplification efficiency, indicating that the method of this invention can rapidly and efficiently amplify the cells.

[0135] Test Example 2

[0136] The cytotoxic activity of γδ T cells was determined using the Cell Counting Kit-8. DU145 prostate cancer cells were used as target cells. Highly active γδ T cells prepared in Examples 4-6 and Comparative Examples 4-10, along with commercially available γδ T cells, were added at an effector-to-target cell ratio of 10:1. The cells were incubated overnight for 16 hours at 37°C with 5% CO2 and 20% oxygen, with the remainder being nitrogen (% is volume percentage). Then, 20 µL / well of CCK-8 was added and incubated for another 3 hours under the same conditions. The absorbance (A) at 450 nm was measured using a microplate reader. After centrifugation at 300 g for 15 min at room temperature, 120 µL of the supernatant was collected, and the IFN-γ content (pg / mL) was determined according to the instructions of the enzyme-linked immunosorbent assay kit at room temperature. The results are shown in Table 2.

[0137] Kill rate (%) = [1 - (OD value of experimental group - OD value of effector cell group alone) / OD value of target cell group alone] × 100%

[0138] Table 2

[0139]

[0140] As shown in the table above, the highly active γδ T cells prepared in Examples 4-6 of this invention have a high killing rate against prostate cancer DU145 cells.

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium, characterized in that, Modified nanoparticles, zoledronic acid, vitamin C, and glutamine were added to the 1640 culture medium, wherein the concentration of the modified nanoparticles was 0.5-1 g / L, the concentration of the zoledronic acid was 0.5-1.5 μmol / L, the concentration of the vitamin C was 200-500 μmol / L, and the concentration of the glutamine was 0.1-0.2 mmol / L. The modified nanoparticles are prepared as follows: S1. Magnetic nanoparticles are added to Tris-HCl solution, dopamine hydrochloride is added, the mixture is heated and stirred to react, separated by magnets, washed, and dried to obtain modified magnetic nanoparticles; the magnetic nanoparticles are iron oxide nanoparticles with a particle size of 20-50 nm. S2. Modified magnetic nanoparticles, zirconium chloride and 1,2,4,5-benzenetetracarboxylic acid were added to water, heated and stirred under reflux, magnetically separated, activated, magnetically separated, washed and dried to obtain UiO-66-(COOH)2@magnetic nanoparticles; S3. Add UiO-66-(COOH)2@ magnetic nanoparticles to water, add NHS and EDC, stir to activate, add disodium pamidronate and alendronate, stir to react, separate with a magnet, wash, and dry to obtain phosphonic acid modified UiO-66-(COOH)2@ magnetic nanoparticles; the mass ratio of UiO-66-(COOH)2@ magnetic nanoparticles, NHS, EDC, disodium pamidronate and alendronate is 12-15:2-4:2-4:1-2:0.8-1.2; S4. Add the interleukin composition to water, add NHS and EDC, stir to activate, add phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles, stir to react, separate with a magnet, wash, dry, and obtain modified nanoparticles; the interleukin composition is a mixture of IL-15, IL-2 and IL-7, and the mass ratio of IL-15, IL-2 and IL-7 in the interleukin composition is 2-3:5-7:1-2.

2. The culture medium according to claim 1, characterized in that, In step S1, the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of the magnetic nanoparticles to dopamine hydrochloride is 10:1-2, and the heating and stirring reaction temperature is 50-60℃ for 3-5 hours.

3. The culture medium according to claim 1, characterized in that, In step S2, the mass ratio of the modified magnetic nanoparticles, zirconium chloride, and 1,2,4,5-benzenetetracarboxylic acid is 10:2-2.5:4-4.

7. The heating and reflux stirring treatment lasts for 20-24 hours. The activation method is to add the nanoparticles to water and heat and reflux for 10-15 hours.

4. The culture medium according to claim 1, characterized in that, The mass ratio of the interleukin composition, NHS, EDC and phosphonic acid modified UiO-66-(COOH)2@magnetic nanoparticles in step S4 is 1-2:0.5-1:0.5-1:10-15.

5. A method for culturing γδ T cells, characterized in that, Includes the following steps: (1) Preparation of peripheral blood human mononuclear cells: peripheral blood was collected, heparin was used for anticoagulation, and human mononuclear cells were separated by density gradient centrifugation with lymphocyte separation solution. The cells were washed with PBS solution and centrifuged to obtain peripheral blood human mononuclear cells. (2) Isolation and purification of γδ T cells: peripheral blood human mononuclear cells were added to the culture medium described in claim 1 and cultured for 1-2 days. Anti-human gamma-delta TCR-FITC antibody-labeled γδ T cells were added, centrifuged, and the culture medium described in claim 1 was added. The γδ T cells were then isolated and purified by flow cytometry. (3) Stimulation of γδ T cells: The isolated and purified γδ T cells were added to the culture medium described in claim 1, the cell density was adjusted, and the cells were treated with low-frequency ultrasound superimposed with a magnetic field. After culturing for 2-4 days, the cells were irradiated with near-infrared light for 20-40 minutes and centrifuged to obtain stimulated γδ T cells. (4) Expansion of γδ T cells: Stimulated γδ T cells are added to the culture medium described in claim 1, the cell density is adjusted, and culture is continued. The medium is replenished every 2-3 days according to the cell density until 12-17 days of culture to obtain highly active γδ T cells.

6. The method for culturing γδ T cells according to claim 5, characterized in that, The step (3) describes adjusting the cell density to 10. 5 -10 6 The low-frequency ultrasonic superimposed magnetic field treatment is performed by superimposing a 10-30mT magnetic field with a 30-50W ultrasonic wave for 1-2 days, and the wavelength of the near-infrared light is 808nm.

7. The method for culturing γδ T cells according to claim 5, characterized in that, The step (4) involves adjusting the cell density to 10. 6 -10 7 Cells / mL, after which the cell density is maintained at 10⁶ / mL. 6 -10 7 per mL.

8. The method for culturing γδ T cells according to claim 5, characterized in that, The culture conditions are 36-38℃, CO2 concentration of 3-7%, oxygen concentration of 25-35%, and the remainder is nitrogen. Here, % refers to volume percentage.

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