A method for preparing gamma delta t cells for clearing tumor cells

Abstract

The application belongs to the technical field of culture medium, and particularly relates to a preparation method of gamma delta T cells for removing tumor cells. The preparation method of the gamma delta T cells for removing tumor cells specifically comprises the following steps: (1) isolating human mononuclear cells PBMCs; (2) inoculating the PBMCs into an anti-CD3 monoclonal antibody coated culture plate for culture, and collecting the cells after culture; (3) adding anti-CD4, anti-CD16 and anti-CD56 antibodies and magnetic beads secondary antibodies to cell suspension to obtain magnetic bead labeled cell suspension; and finally obtaining gamma delta T cells; and (4) inoculating the gamma delta T cells into a proliferation culture medium for culture, and obtaining the gamma delta T cells. The application provides the preparation method of the gamma delta T cells, realizes efficient expansion of the gamma delta T cells, significantly improves tumor killing activity of the gamma delta T cells, provides a high-quality culture scheme for clinical application of the cells, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of culture medium technology, specifically relating to a method for preparing γδT cells for eliminating tumor cells. Background Technology

[0002] In recent years, cell immunotherapy has become a crucial pillar in the field of cancer treatment. Among them, T-cell-based cell therapies, especially genetically engineered T-cell therapies represented by CAR-T, have achieved groundbreaking progress in the treatment of hematological malignancies. However, this type of therapy still faces many challenges, including limited efficacy against solid tumors, high costs and long production cycles due to reliance on personalized gene editing, and potential serious side effects such as cytokine release syndrome.

[0003] Among the many T cell subsets, γδT cells have attracted much attention due to their unique biological characteristics. Unlike the dominant αβT cells, γδT cell activation does not depend on antigen presentation via the major histocompatibility complex (MHC), enabling them to directly recognize and kill a wide range of tumor cells, especially exhibiting a natural killing advantage against immune-evading tumor cells with low or absent MHC molecules. This makes γδT cells a potentially versatile "off-the-shelf" cell therapy product.

[0004] However, key bottlenecks remain in translating γδT cells into effective clinical therapies: First, the challenge of expansion. γδT cells are present in extremely low concentrations in human peripheral blood (accounting for only 2-5% of T cells), and there is a lack of efficient and stable in vitro expansion protocols to obtain the large number of cells required for treatment. Second, the challenge of maintaining function. During in vitro expansion, γδT cells are prone to functional exhaustion or apoptosis, leading to decreased in vivo persistence and antitumor activity. Third, deficiencies in culture systems. Existing culture methods often rely on heterologous serum, feeder cells, or large amounts of cytokines, resulting in unclear composition, significant batch-to-batch variability, risks associated with introducing exogenous substances, and high costs, severely hindering their clinical translation and large-scale production.

[0005] Therefore, developing a method for preparing γδT cells that is well-defined in composition, highly efficient and stable, and capable of simultaneously achieving large-scale expansion of γδT cells while maintaining their potent killing function has become a core technical problem urgently needing to be solved in this field. To overcome these challenges, this invention provides a method for preparing γδT cells for eliminating tumor cells. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for preparing γδT cells for eliminating tumor cells.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing γδT cells for eliminating tumor cells specifically includes the following steps:

[0009] (1) Collect peripheral blood samples, separate peripheral blood mononuclear cells, resuspend, wash, and centrifuge to finally obtain PBMCs;

[0010] (2) The PBMCs obtained in step (1) were resuspended in basal medium, seeded in culture plates coated with anti-CD3 monoclonal antibody, and the cultured cells were collected.

[0011] (3) The cells obtained in step (2) were resuspended in a cell suspension with buffer, and anti-CD4 antibody, anti-CD16 antibody and anti-CD56 antibody were added and incubated to obtain an antibody-labeled mixture; then magnetic beads were added and incubated to obtain a magnetic bead-labeled cell suspension; the magnetic bead-labeled cell suspension was sorted and centrifuged to obtain γδT cells;

[0012] (4) The γδT cells obtained in step (3) are seeded into a proliferation medium and cultured to obtain a γδT cell for clearing tumor cells; the proliferation medium includes a basal medium and the following components added to the basal medium: transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide; the basal medium is serum-free RPMI 1640 basal medium.

[0013] Further, based on the final concentration, the concentration of transferrin in the proliferation medium of step (4) is 5-15 mg / L, the concentration of insulin is 5-15 mg / L, the concentration of IL-2 is 5-10 ng / mL, the concentration of vitamin C is 0.5-5 µg / mL, the concentration of L-arginine-α-ketoglutarate is 0.4-2.5 mg / mL, and the concentration of metoclopramide is 15-25 ng / mL.

[0014] Further, based on the final concentration, the concentration of transferrin in the proliferation medium of step (4) is 10 mg / L, the concentration of insulin is 10 mg / L, the concentration of IL-2 is 7 ng / mL, the concentration of vitamin C is 2.5 µg / mL, the concentration of L-arginine-α-ketoglutarate is 1.4 mg / mL, and the concentration of metoclopramide is 20 ng / mL.

[0015] Further, the basal culture medium in step (2) is RPMI 1640 basal culture medium containing 10 v / v% FBS; the inoculation density is 2-4 × 10⁻⁴. 6 The concentration of anti-CD3 monoclonal antibody was 1-3 μg / mL; the culture conditions were 37℃, 5v / v%CO2; and the culture time was 3-5 days.

[0016] Further, based on the final concentration, the concentration of anti-CD4 antibody in the antibody-labeled mixture in step (3) is 0.5-1 μg / mL; the concentration of anti-CD16 antibody is 1-2 μg / mL; the concentration of anti-CD56 antibody is 0.5-1.5 μg / mL; the incubation time is 10-20 min; and the incubation time of magnetic beads is 15-25 min.

[0017] Further, the specific operation of step (4) is as follows: the γδT cells obtained in step (3) are processed at a rate of 1-3 × 10⁻⁶. 6 The cells were seeded at a density of 1 cell / mL into proliferation medium and cultured at 37°C with 5 v / v% CO2. The medium was then changed halfway every 2-3 days, and the cells were cultured for 10-15 days to obtain a type of γδT cells for clearing tumor cells.

[0018] Compared with the prior art, the main advantages of the present invention are as follows:

[0019] This invention provides a method for preparing γδT cells for eliminating tumor cells. By adding L-arginine-α-ketoglutarate and metoclopramide to the proliferation medium, the tumor-killing activity of γδT cells is significantly enhanced while achieving efficient expansion. Furthermore, the proliferation medium has a clear and simple composition, readily available raw materials, high stability, requires no antibiotics, and contains no animal-derived components, thus solving the toxic side effects of animal-derived culture components on cell physiological functions. This provides a high-quality culture protocol for clinical cell applications and has broad application prospects. Attached Figure Description

[0020] Figure 1 This is a flowchart of a method for preparing γδT cells for eliminating tumor cells according to the present invention;

[0021] Figure 2 This is a morphological diagram of γδT cells obtained in Example 1 of the present invention;

[0022] Figure 3 This is a morphological image of γδT cells obtained in Example 2 of the present invention;

[0023] Figure 4 This is a morphological diagram of γδT cells obtained in Example 3 of the present invention.

[0024] Figure 5 This is a morphological diagram of γδT cells obtained from Comparative Example 1 of this invention.

[0025] Figure 6 This is a morphological diagram of γδT cells obtained from Comparative Example 2 of this invention.

[0026] Figure 7 The killing rate of γδT cells against tumor cells. Detailed Implementation

[0027] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0028] Example 1

[0029] A method for preparing γδT cells for eliminating tumor cells specifically includes the following steps:

[0030] (1) Collect peripheral blood samples, separate peripheral blood mononuclear cells, resuspend, wash, and centrifuge to finally obtain PBMCs;

[0031] (2) The PBMCs obtained in step (1) were resuspended in RPMI 1640 basal medium containing 10 v / v% FBS, and then subjected to a reaction at 3 × 10⁻⁶ ppm. 6 Cells were seeded at a density of 1 cell / mL in culture plates coated with 2 μg / mL anti-CD3 monoclonal antibody and cultured for 4 days at 37°C and 5 v / v % CO2. The cultured cells were then collected.

[0032] (3) Resuspend the cells obtained in step (2) in pre-cooled cell sorting buffer to a cell suspension, add anti-CD4 antibody, anti-CD16 antibody, and anti-CD56 antibody, and incubate at 4°C for 15 min to obtain an antibody-labeled mixture. The final concentrations of the antibody-labeled mixture are: anti-CD4 antibody 0.7 μg / mL; anti-CD16 antibody 1.5 μg / mL; and anti-CD56 antibody 1.0 μg / mL. After washing with buffer, [the mixture is then] 1×10 [units of solution]. 7 20 µL of magnetic bead secondary antibody was added to each cell, and the cells were incubated at 4 °C for 20 min to obtain a magnetic bead-labeled cell suspension. The magnetic bead-labeled cell suspension was loaded onto a sorting column on a magnetic rack, the flow-through was collected, and the cells were centrifuged to obtain γδT cells.

[0033] (4) The γδT cells obtained in step (3) were used at a rate of 2×10 6The cells were seeded at a density of [number] cells / mL in proliferation medium and cultured at 37°C with 5 v / v% CO2. The medium was then changed halfway every 2 days, and the cells were cultured for 12 days to obtain a type of γδT cells for eliminating tumor cells. The proliferation medium consisted of serum-free RPMI 1640 basal medium, and the following components were added to this basal medium: transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide. The final concentrations of transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide in the proliferation medium were 10 mg / L, 10 mg / L, 7 ng / mL, 2.5 µg / mL, 1.4 mg / mL, and 20 ng / mL, respectively. A flowchart of a method for preparing γδT cells for eliminating tumor cells is shown below. Figure 1 As shown.

[0034] Example 2

[0035] A method for preparing γδT cells for eliminating tumor cells specifically includes the following steps:

[0036] (1) Collect peripheral blood samples, separate peripheral blood mononuclear cells, resuspend, wash, and centrifuge to finally obtain PBMCs;

[0037] (2) The PBMCs obtained in step (1) were resuspended in RPMI 1640 basal medium containing 10 v / v% FBS, and then thawed at 2 × 10⁻⁶ ppm. 6 Cells were seeded at a density of 1 μg / mL in culture plates coated with anti-CD3 monoclonal antibody and cultured for 3 days at 37°C and 5 v / v % CO2. The cultured cells were then collected.

[0038] (3) Resuspend the cells obtained in step (2) in pre-cooled cell sorting buffer to a cell suspension, add anti-CD4 antibody, anti-CD16 antibody, and anti-CD56 antibody, and incubate at 4°C for 10 min to obtain an antibody-labeled mixture. The final concentrations of the antibody-labeled mixture are: anti-CD4 antibody 0.5 μg / mL; anti-CD16 antibody 1 μg / mL; and anti-CD56 antibody 0.5 μg / mL. After washing with buffer, proceed with 1×10⁻⁶ cells. 7 Add 20 µL of magnetic bead secondary antibody to each cell and incubate at 4 °C for 15 min to obtain a magnetic bead-labeled cell suspension. Load the magnetic bead-labeled cell suspension onto a sorting column on a magnetic rack, collect the flow-through, and centrifuge to obtain γδT cells.

[0039] (4) The γδT cells obtained in step (3) were used at a concentration of 1×10⁻⁶. 6γδT cells were seeded at a density of 1 / mL into proliferation medium and cultured at 37°C with 5 v / v% CO2. The medium was then changed every 2-3 days for 10 days to obtain a type of γδT cells for eliminating tumor cells. The proliferation medium consisted of serum-free RPMI 1640 basal medium, with the following components added to it: transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide. The final concentrations of transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide in the proliferation medium were 5 mg / L, 15 mg / L, 5 ng / mL, 0.5 µg / mL, 0.4 mg / mL, and 15 ng / mL, respectively.

[0040] Example 3

[0041] A method for preparing γδT cells for eliminating tumor cells specifically includes the following steps:

[0042] (1) Collect peripheral blood samples, separate peripheral blood mononuclear cells, resuspend, wash, and centrifuge to finally obtain PBMCs;

[0043] (2) The PBMCs obtained in step (1) were resuspended in RPMI 1640 basal medium containing 10 v / v% FBS, and then subjected to a reaction at 4 × 10⁻⁶ ppm. 6 Cells were seeded at a density of 1 cell / mL in culture plates coated with 3 μg / mL anti-CD3 monoclonal antibody and cultured for 5 days at 37°C and 5 v / v % CO2. The cultured cells were then collected.

[0044] (3) Resuspend the cells obtained in step (2) in pre-cooled cell sorting buffer to a cell suspension, add anti-CD4 antibody, anti-CD16 antibody, and anti-CD56 antibody, and incubate at 4°C for 20 min to obtain an antibody-labeled mixture. The final concentrations of the antibody-labeled mixture are: anti-CD4 antibody 1 μg / mL; anti-CD16 antibody 2 μg / mL; and anti-CD56 antibody 1.5 μg / mL. After washing with buffer, proceed with 1×10⁻⁶ cells. 7 20 µL of magnetic bead secondary antibody was added to each cell, and the cells were incubated at 4 °C for 25 min to obtain a magnetic bead-labeled cell suspension. The magnetic bead-labeled cell suspension was loaded onto a sorting column on a magnetic rack, the flow-through was collected, and the cells were centrifuged to obtain γδT cells.

[0045] (4) The γδT cells obtained in step (3) were used in a process of 3×10 6γδT cells were seeded at a density of 1 / mL into proliferation medium and cultured at 37°C with 5% v / v CO2. The medium was then changed halfway every 3 days, and the culture was repeated for 15 days to obtain a type of γδT cells for eliminating tumor cells. The proliferation medium consisted of serum-free RPMI 1640 basal medium, with the following components added to this basal medium: transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide. The final concentrations of transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide in the proliferation medium were 15 mg / L, 15 mg / L, 10 ng / mL, 5 µg / mL, 2.5 mg / mL, and 25 ng / mL, respectively.

[0046] Comparative Example 1

[0047] The difference between Comparative Example 1 and Example 1 is that L-arginine-α-ketoglutarate in the proliferation culture medium was omitted, while all other components were the same as in Example 1.

[0048] Comparative Example 2

[0049] The difference between Comparative Example 2 and Example 1 is that metoclopramide in the proliferation culture medium was omitted, while everything else was the same as in Example 1.

[0050] Example 1

[0051] γδT cell viability assay:

[0052] A type of γδT cells for eliminating tumor cells was prepared using the methods described in Examples 1-3, Comparative Examples 1 and 2. The morphology of the γδT cells after 10 days of expansion and culture in Examples 1-3, Comparative Examples 1 and 2 is as follows. Figure 2-6 As shown in Table 1, the viability of γδT cells from Examples 1-3, Comparative Examples 1, and Comparative Examples 2 after 10 days of amplification culture in step (4) was detected using trypan blue staining. The specific steps are as follows: γδT cells prepared in Examples 1-3, Comparative Examples 1, and Comparative Examples 2 were mixed with 0.4% trypan blue solution at a 1:1 ratio, allowed to stand for 2 minutes, and counted using an automated cell counter. Live cells were colorless and transparent, while dead cells were blue. Cell viability (%) = [(number of live cells) / (number of live cells + number of dead cells)] × 100%. The results are shown in Table 1.

[0053] Table 1. Cell viability (%)

[0054]

[0055] As shown in Table 1, compared with Comparative Examples 1 and 2, the γδT cells cultured in Examples 1-3 of this invention exhibited higher cell viability, exceeding 94%. Among them, the γδT cells prepared using the method in Example 1 showed the strongest cell viability. This indicates that L-arginine-α-ketoglutarate and metoclopramide in the proliferation medium significantly improved the viability of γδT cells. L-arginine, as a key amino acid for T cell proliferation, and α-ketoglutarate, as a key intermediate in the tricarboxylic acid cycle, provide crucial metabolic substrates, jointly promoting healthy cell metabolism. Metoclopramide may enhance cell viability by regulating neurotransmitter receptors and influencing cellular stress responses or microenvironmental signals. Both together create a more favorable environment for the survival and proliferation of γδT cells, thus affecting cell viability.

[0056] Experimental Example 2

[0057] γδT cell killing rate against tumor cells

[0058] To detect the cytotoxic activity of γδT cells obtained in Examples 1-3 and Comparative Examples 1 and 2 against prostate cancer DU145 cells, γδT cells obtained in Examples 1-3 and Comparative Examples 1 and 2, as well as commercially available γδT cells, were used as effector cells, and prostate cancer DU145 cells were used as target cells. Effector cells and target cells were added to 96-well plates at an effector-to-target ratio of 10:1 to form experimental groups, with three replicates per gradient. Effector cells were mixed thoroughly with RPMI-1640 medium to form the effector cell group alone, used to assess the proliferation or activity of γδT cells. Target cells were mixed thoroughly with RPMI-1640 medium to form the target cell group alone, used to assess the growth of DU145 cells in the absence of γδT cells. The 96-well plates were incubated overnight for 10 hours at 37°C in an incubator with 5 v / v% CO2 and 20 v / v% oxygen, with the balance being nitrogen. 20 μL of CCK-8 solution was added to each well according to the CCK-8 kit instructions. Continue culturing for 4 hours, and use a microplate reader to measure the absorbance of each well at a wavelength of 490 nm. Calculate the killing rate based on the absorbance value: Cell killing rate % = [1 - (OD value of experimental group - OD value of effector cell group alone) / (OD value of target cell group alone)] × 100%.

[0059] The results are as follows Figure 7 The figure shows the killing rate of γδT cells against tumor cells. (From...) Figure 7It can be seen that, compared with Comparative Examples 1 and 2, and the commercially available γδT cell group, the γδT cells prepared in Examples 1-3 of this invention can significantly improve the killing rate of γδT cells against DU145 cells. At an effector-to-target ratio of 10:1, the killing rate of γδT cells prepared in Example 1 reached as high as 88.9%. It is speculated that the L-arginine-α-ketoglutarate and metoclopramide components in the proliferation culture medium can enhance the activity of γδT cells, thereby increasing the tumor-killing activity of γδT cells and improving the cell killing rate.

[0060] Further analysis revealed that, compared to Example 1, Comparative Example 1 omitted L-arginine-α-ketoglutarate from its proliferation medium, and Comparative Example 2 omitted metoclopramide from its proliferation medium. Both groups showed a significant decrease in the proliferation capacity and tumor killing rate of γδT cells. This indicates that L-arginine-α-ketoglutarate and metoclopramide in the proliferation medium can effectively enhance the proliferation capacity of γδT cells and improve their antitumor activity.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A method for preparing γδT cells for eliminating tumor cells, characterized in that, Specifically, the following steps are included: (1) Collect peripheral blood samples, separate peripheral blood mononuclear cells, resuspend, wash, and centrifuge to finally obtain PBMCs; (2) The PBMCs obtained in step (1) were resuspended in basal medium, seeded in culture plates coated with anti-CD3 monoclonal antibody, and the cultured cells were collected. (3) The cells obtained in step (2) were resuspended in a cell suspension with buffer, and anti-CD4 antibody, anti-CD16 antibody and anti-CD56 antibody were added and incubated to obtain an antibody-labeled mixture; then magnetic beads were added and incubated to obtain a magnetic bead-labeled cell suspension; the magnetic bead-labeled cell suspension was sorted and centrifuged to obtain γδT cells; (4) The γδT cells obtained in step (3) are seeded into a proliferation medium and cultured to obtain a γδT cell for clearing tumor cells; the proliferation medium includes a basal medium and the following components added to the basal medium: transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide; the basal medium in step (4) is serum-free RPMI 1640 basal medium.

2. The method for preparing γδT cells for eliminating tumor cells according to claim 1, characterized in that, Based on the final concentration, the concentrations of transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide in the proliferation medium of step (4) are 5-15 mg / L, 5-15 mg / L, 5-10 ng / mL, 0.5-5 µg / mL, 0.4-2.5 mg / mL, and 15-25 ng / mL, respectively.

3. The method for preparing γδT cells for eliminating tumor cells according to claim 2, characterized in that, Based on the final concentration, the concentrations of transferrin, insulin, IL-2, vitamin C, L-arginine-α-ketoglutarate, and metoclopramide in the proliferation medium of step (4) are 10 mg / L, 10 mg / L, 7 ng / mL, 2.5 µg / mL, 1.4 mg / mL, and 20 ng / mL, respectively.

4. The method for preparing γδT cells for eliminating tumor cells according to claim 1, characterized in that, The basal culture medium in step (2) is RPMI 1640 basal culture medium containing 10 v / v% FBS; the inoculation density is 2-4 × 10⁻⁴. 6 The concentration of anti-CD3 monoclonal antibody was 1-3 μg / mL; the culture conditions were 37℃, 5v / v%CO2; and the culture time was 3-5 days.

5. The method for preparing γδT cells for eliminating tumor cells according to claim 1, characterized in that, Based on the final concentration, the concentration of anti-CD4 antibody in the antibody-labeled mixture in step (3) is 0.5-1 μg / mL; the concentration of anti-CD16 antibody is 1-2 μg / mL; the concentration of anti-CD56 antibody is 0.5-1.5 μg / mL; the incubation time is 10-20 min; and the incubation time of magnetic beads is 15-25 min.

6. The method for preparing γδT cells for eliminating tumor cells according to claim 1, characterized in that, The specific operation of step (4) is as follows: the γδT cells obtained in step (3) are processed at a rate of 1-3 × 10⁻⁶. 6 The cells were seeded at a density of 1 cell / mL into proliferation medium and cultured at 37°C with 5 v / v% CO2. The medium was then changed halfway every 2-3 days, and the cells were cultured for 10-15 days to obtain a type of γδT cells for clearing tumor cells.

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