Immune cell culture kit, culture method of gamma delta t cells and application

Abstract

The application provides an immune cell culture kit, a culture method and application of gamma delta T cells, and the kit comprises a basic culture medium, a recombinant antibody, a cytokine and a cell proliferation promoting additive, the basic culture medium comprises an RPMI-1640 culture medium, and the cell proliferation promoting additive comprises 1-thioglycerol, taurine, beta-cyclodextrin, thymidine, alpha-lipoic acid, beta-mercaptoethanol and bisphosphonate. The gamma delta T cells obtained by the culture kit have high purity and activity, and have good tumor killing activity. The gamma delta T cells obtained by the method have a high positive rate of 100% of perforin expression.

Description

Technical Field

[0001] This invention relates to the field of γδT cell culture technology, and in particular to an immune cell culture kit, a γδT cell culture method, and its application. Background Technology

[0002] Immune cells are the basic components of the body's immune system. They can recognize and eliminate pathogens that invade the body, including lymphocytes and macrophages, and play a vital role in the human immune system.

[0003] γδT cells are a special type of T cell in the immune system, mainly distributed in the mucous membranes and subcutaneous tissues of the intestines, respiratory tract, and genitourinary tract. They are one of the main components of intraepithelial lymphocytes (IELs) in mucosal tissues. In healthy adults, γδT cells account for 1% to 5% of peripheral blood T cells. However, γδT cells can directly kill cancer cells and tumor stem cells, recognize carcinoembryonic antigens, and also directly kill tumor cells through perforin granzyme pathways.

[0004] γδT cell therapy has shown great potential in cancer treatment. Although γδT cell immunotherapy has shown great application potential, the current culture of γδT cells has the problem of low expansion rate. How to improve the expansion rate of γδT cells is an urgent problem to be solved. Summary of the Invention

[0005] This invention provides an immune cell culture kit, a method for culturing γδT cells, and their applications. The main objective of this invention is to solve the technical problems mentioned in the background section of the prior art.

[0006] The contents of this invention are as follows:

[0007] The first aspect of the present invention provides an immune cell culture kit, comprising: a basal culture medium, a recombinant antibody, cytokines and cell proliferation promoters, wherein the basal culture medium comprises RPMI-1640 medium, and the cell proliferation promoters comprise 1-thioglycerol, taurine, β-cyclodextrin, thymidine, α-lipoic acid, β-mercaptoethanol and bisphosphonates.

[0008] In this invention, the immune cell culture kit consists of components A, B, C, and D, wherein component A is the basal culture medium, and the basal culture medium used in this invention is RPMI-1640; component B is a recombinant antibody; component C is a cell proliferation promoter; and component D is a cytokine.

[0009] Among them, recombinant antibodies are human recombinant antibodies used to activate corresponding cells and promote cell proliferation, as well as enhance the immune response of target cells. Cytokines are used to promote cell expansion, activation, and maturation. The addition of cell proliferation-promoting additives can further promote cell proliferation, increase its expansion rate and cell survival rate, and provide more in-depth support for cell culture.

[0010] In this invention, the cell proliferation-promoting additive used is a combination of 1-thioglycerol, taurine, β-cyclodextrin, thymidine, α-lipoic acid, β-mercaptoethanol, and bisphosphonates. This cell proliferation-promoting additive is combined with the RPMI-1640 basal culture medium, along with cytokines and recombinant antibodies, to improve cell survival rate and proliferation rate.

[0011] In an optional embodiment of the first aspect of the present invention, the bisphosphate is one or both of pamidronate disodium and zoledronic acid.

[0012] In this invention, the bisphosphates in the cell proliferation-promoting additives are pamidronate disodium and / or zoledronic acid, which have a significant stimulatory effect on the proliferation of γδT cells cultured in PBMCs. By expanding γδT cells, pamidronate disodium and / or zoledronic acid help enhance the body's immune response, thereby playing an important role in the treatment of diseases such as tumors and infections.

[0013] Specifically, in the cell proliferation-promoting additive, the bisphosphate can be 1-10 mg / mL pamidronate disodium and / or 1-10 mg / mL zoledronic acid. In one specific embodiment, the bisphosphate is 1-10 mg / mL pamidronate disodium.

[0014] In an optional embodiment of the first aspect of the present invention, the recombinant antibody is one or both of anti-human TCR V delta 1 monoclonal antibody and anti-human TCR V delta 2 monoclonal antibody (15D). The selected recombinant antibody is an anti-human TCR V delta 1 monoclonal antibody with clone number TS8.2, and / or an anti-human TCR V delta 2 monoclonal antibody (15D), wherein TCR V delta 1 and TCR V delta 2 are both important markers on the surface of γδT cells, participating in multiple stages of the immune response, and facilitating subsequent detection and separation. Specifically, in a preferred embodiment, the recombinant antibody is a combination of 25–200 μg / mL anti-human TCR V delta 1 monoclonal antibody (TS8.2) and 25–200 μg / mL anti-human TCR V delta 2 monoclonal antibody (15D).

[0015] In an optional embodiment of the first aspect of the present invention, the cytokines include 500,000 to 1,000,000 U / mL IL-2, 10 to 100 μg / mL IL-15, and 1 to 10 μg / mL IL-21.

[0016] In an optional embodiment of the first aspect of the present invention, in the cell proliferation-promoting additive, the concentration of 1-thioglycerol is 1-10 mg / mL, the concentration of taurine is 1-10 mg / mL, the concentration of β-cyclodextrin is 1-10 mg / mL, the concentration of thymidine is 1-10 mg / mL, the concentration of α-lipoic acid is 1-10 mg / mL, and the concentration of β-mercaptoethanol is 1-10 mM.

[0017] As a second aspect of the present invention, the present invention also provides a method for culturing γδT cells, comprising:

[0018] Provide an immune cell culture kit as described in the first aspect above;

[0019] The culture vessel was coated with a mixture of recombinant antibody and buffer; the basal culture medium, cytokines and cell proliferation promoters were mixed to obtain the first mixed culture medium;

[0020] PBMCs were isolated from human peripheral blood.

[0021] PBMCs were seeded into coated culture containers, and cultured in the first mixed medium for a predetermined time. During the culture process, the first mixed medium was supplemented to maintain a cell concentration of 2.0–3.5 × 10⁻⁶ cells / year. 6 cells / mL;

[0022] Cells collected from the culture container after the predetermined culture time.

[0023] The culture containers referred to in this invention include culture flasks or culture bags, such as T25 or T75 culture flasks, T175 culture flasks, T225 culture flasks, and 1L culture bags, etc. Depending on the actual volume of the culture medium, they can be transferred or replaced to ensure sufficient space for culture.

[0024] In the γδT cell culture method, firstly, the culture vessel is coated with a mixture of recombinant antibody and buffer solution. The buffer solution used is D-PBS buffer, and the volume ratio of recombinant antibody to the buffer solution is 1:9. Specifically, during the coating process, a mixture of recombinant antibody component and 9 mL of D-PBS buffer (1:9) is used to coat T25 or T75 culture flasks at room temperature for 1 hour or at 4°C overnight. After coating, the coating solution is discarded, and the culture flask is washed twice with PBS for later use.

[0025] During cultivation, PBMCs are inoculated into coated culture containers at an inoculation density of 3–8 × 10⁻⁶. 6 per mL.

[0026] In an optional embodiment of the second aspect of the present invention, the method further includes adding autologous plasma to the culture container during the initial stage of culture, wherein the concentration of the autologous plasma is 1-10%, and the initial stage of culture is 0-9 days of a predetermined time.

[0027] Specifically, the entire culture cycle is 14 days. During the first 9 days of culture, 1-10% autologous plasma can be added, preferably at a concentration of 10%. After 9 days, no further addition of autologous plasma is needed. The supplementation of autologous plasma can meet the nutritional needs of cell culture and help cells grow and proliferate in the culture environment.

[0028] In an optional embodiment of the second aspect of the present invention, the first mixed culture medium is obtained by mixing basal culture medium: cytokines: cell proliferation-promoting additives in a ratio of 1000:1:1.

[0029] In a third aspect of the invention, an application is provided of the immune cell culture kit as described above in the preparation of immune cells and / or supernatant exosomes.

[0030] The fourth aspect of the present invention provides the use of γδT cells obtained as described above in the preparation of drugs for treating and / or preventing tumors.

[0031] Beneficial Effects: This invention provides an immune cell culture kit, culture method, and application. The kit includes a basal culture medium, recombinant antibody, cytokines, and cell proliferation-promoting additives. The basal culture medium includes RPMI-1640 medium, and the cell proliferation-promoting additives include 1-thioglycerol, taurine, β-cyclodextrin, thymidine, α-lipoic acid, β-mercaptoethanol, and bisphosphonates. The γδT cells cultured using this invention's kit have high purity and viability, and exhibit good tumor-killing activity. The γδT cells cultured using this method show a 100% positive rate of perforin expression. Compared to existing culture methods, the γδT cells obtained by the culture method of this invention have higher cell viability, proliferation rate, purity, and killing activity. Attached Figure Description

[0032] Figure 1 Schematic diagram showing the effect of different concentrations of pamidronate disodium on the proliferation of γδT cells;

[0033] Figure 2 Schematic diagram showing the effect of different concentrations of zoledronic acid on the proliferation of γδT cells;

[0034] Figure 3 A schematic diagram showing the effects of different concentrations of anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and anti-human TCR V delta 2 Monoclonal Antibody (15D) on the proliferation of γδT cells;

[0035] Figure 4 This is a schematic diagram showing the fold expansion of γδT cells in Example 1 and Comparative Example 1;

[0036] Figure 5 This is a flow cytometry diagram illustrating the purity of γδT cells in Example 1 and Comparative Example 1.

[0037] Figure 6 This is a flow cytometry diagram of perforin expression in γδT cells in Example 1 and Comparative Example 1. Detailed Implementation

[0038] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0039] As a first aspect of the present invention, the immune cell kit of the present invention comprises a culture medium kit consisting of components A, B, C, and D:

[0040] Component A is the basal culture medium, including RPMI-1640;

[0041] Component B consists of human recombinant antibodies, including 25–200 μg / mL anti-human TCR V delta1 Monoclonal Antibody (TS8.2) and / or 25–200 μg / mL anti-human TCR V delta2 Monoclonal Antibody (15D);

[0042] Component C consists of cell proliferation-promoting additives, including 1–10 mg / mL 1-thioglycerol, 1–10 mg / mL taurine, 1–10 mg / mL β-cyclodextrin, 1–10 mg / mL thymidine, 1–10 mg / mL α-lipoic acid, 1–10 mM β-mercaptoethanol, and 1–10 mg / mL bisphosphonates.

[0043] Component D consists of cytokines, including: 500,000–1,000,000 U / mL IL-2, 10–100 μg / mL IL-15, and 1–10 μg / mL IL-21.

[0044] Specifically, in component B, the human recombinant antibody can be one or both of anti-human TCR V delta1 Monoclonal Antibody (TS8.2) and anti-human TCR V delta2 Monoclonal Antibody (15D). For example, it can be only anti-human TCR V delta1 Monoclonal Antibody (TS8.2) at a concentration of 25-200 μg / mL. The 25-200 μg / mL refers to any concentration within the range, such as 25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 180 μg / mL, or 200 μg / mL. It can also be a combination of the two. In the preferred embodiment of the present invention, component B includes 100 μg / mL anti-human TCR V delta1 Monoclonal Antibody (TS8.2) and 100 μg / mL anti-human TCR V delta2 Monoclonal Antibody (15D).

[0045] Specifically, the bisphosphate includes one or both of pamidronate disodium and zoledronic acid, preferably pamidronate disodium.

[0046] In a preferred embodiment, the immune cell kit comprises a culture medium kit, wherein component B consists of human recombinant antibodies including 100 μg / mL anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and 100 μg / mL anti-human TCR V delta 2 Monoclonal Antibody (15D); component C consists of 6 mg / mL 1-thioglycerol, 4 mg / mL taurine, 5 mg / mL β-cyclodextrin, 8 mg / mL thymidine, 2 mg / mL α-lipoic acid, 5 mM β-mercaptoethanol, and 5 mg / mL pamidronate disodium; and component D consists of 700,000 U / mL IL-2, 50 μg / mL IL-15, and 8 μg / mL IL-21.

[0047] The kit of this invention uses RPMI-1640 as the basal culture medium, to which cell proliferation promoters are added, including 1-thioglycerol, taurine, β-cyclodextrin, thymidine, α-lipoic acid, β-mercaptoethanol, and bisphosphate. Taurine, as an amino acid, is essential for cellular protein synthesis and metabolism. In PBMC culture, taurine may act as a nutrient, promoting the proliferation and differentiation of γδT cells, while also increasing their cytotoxicity. The synergistic effect of these components regulates a more suitable environment for γδT cell survival, thereby improving their survival rate and expansion rate, and simultaneously enhancing their antitumor activity.

[0048] A second aspect of the invention claims protection for the application of the immune cell culture kit in the in vitro preparation of γδT cells, specifically, the use of the immune cell culture kit to culture γδT cells in PBMCs, resulting in γδT cells with high survival and expansion rates.

[0049] In a second aspect, the invention seeks protection for the application of the above-mentioned immune cell culture kit in immune cell amplification and immune cell exosome preparation. The immune cell culture kit can improve the amplification rate of immune cells in vitro, and the cost of the immune cell culture kit is relatively low, which helps enterprises save costs.

[0050] In a fourth aspect of the present invention, the γδT cells obtained in the second aspect of the present invention are used in the preparation of some tumor drugs. The obtained γδT cells have a perforin expression positive rate of up to 100%, indicating that they have stronger killing activity.

[0051] To better illustrate the technical effects of the present invention, the present invention has constructed the following embodiments and comparative examples for testing.

[0052] Example 1: The immune cell culture kit used consists of components A, B, C, and D:

[0053] Component A is the basal culture medium, 2 × 1 L, including RPMI-1640;

[0054] Component B is a human recombinant antibody, 1 mL, including 100 μg / mL anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and 100 μg / mL anti-human TCR V delta 2 Monoclonal Antibody (15D);

[0055] Component C is a cell proliferation-promoting additive, 2×1mL, 6mg / mL 1-thioglycerol, 4mg / mL taurine, 5mg / mL β-cyclodextrin, 8mg / mL thymidine, 2mg / mL α-lipoic acid, 5mM β-mercaptoethanol, and 5mg / mL disodium pamidronate.

[0056] Component D consists of cytokines, 2 × 1 mL, including: 700,000 U / mL IL-2, 50 μg / mL IL-15, and 8 μg / mL IL-21.

[0057] The culturing steps for the γδT cells are as follows:

[0058] S1: Coat a T25 or T75 culture flask with a mixture of 1 mL of component B and 9 mL of D-PBS at room temperature for 1 hour or overnight at 4°C. After coating, discard the coating solution and wash the culture flask twice with PBS for later use.

[0059] S2: Isolation of human peripheral blood mononuclear cells (PBMCs);

[0060] The separation method in step S2 is the Ficol density gradient centrifugation method.

[0061] S3: Preparation of mixed culture medium: Prepare 2L of mixed culture medium by mixing component A: component C: component D = 1000: 1: 1 to obtain the first mixed culture medium;

[0062] S4: Inoculate PBMCs into coated T25 or T75 culture flasks at a density of 3–8 × 10⁶. 6 The cells / mL were added and cultured with an appropriate amount of the first mixed culture medium.

[0063] S5: Nine days before culture, add 10% autologous plasma to the culture system. Simultaneously, during culture, replenish the first mixed culture medium as needed based on cell density and medium color, maintaining the cell concentration at 2.0–3.5 × 10⁻⁶ cells / day.6 Following the principle of cells / mL, cells were sequentially transferred to T175 culture flasks, T225 culture flasks, and 1L culture bags according to the volume of culture medium, until day 14 of culture.

[0064] S6: After culture, cells were collected and tested for γδT cell viability, purity, expansion fold, and perforin expression, a marker of cytotoxic activity.

[0065] Controlled test experiment

[0066] The effects of different concentrations of pamidronate disodium and zoledronic acid on the proliferation of γδT cells were determined using the controlled variable method.

[0067] Specifically, the immune cell culture kit for control test group 1 was the same as that in Example 1, with the contents of other components kept constant. Different concentrations of pamidronate disodium were set, namely 0, 0.5 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. The culture method was the same as in Example 1. The total number of cells after 14 days of culture was then detected, and the amplification fold was calculated.

[0068] Specifically, the immune cell culture kit for control test group 2 consisted of replacing pamidronate disodium with zoledronic acid, with the remaining components and concentrations the same as in Example 1. Different concentrations of zoledronic acid were set, corresponding to 0, 0.5 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively. The culture method was the same as in Example 1. The total number of cells after 14 days of culture was then detected, and the amplification fold was calculated.

[0069] like Figure 1 and Figure 2 As shown, Figure 1 This serves as a control for the fold increase in γδT cell proliferation at different concentrations of pamidronate disodium. Figure 2 As a control for the fold increase in γδT cell proliferation at different concentrations of zoledronic acid, from... Figure 1 and Figure 2 It can be observed that the fold increase in γδT cell expansion increases with increasing concentrations of pamidronate disodium and zoledronic acid. However, at a concentration of 20 μg / mL, the fold increase in γδT cell expansion is lower than that at 10 μg / mL. This indicates that high concentrations of pamidronate disodium and / or zoledronic acid can over-activate γδT cells and induce apoptosis. Therefore, the amount of bisphosphate added must be controlled within the range defined in this invention. Furthermore, the fold increase of 5 μg / mL pamidronate and 10 μg / mL zoledronic acid is comparable, therefore pamidronate is preferred as the bisphosphate.

[0070] The effects of different concentrations of anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and anti-human TCR V delta 2 Monoclonal Antibody (15D) on γδT cell proliferation were measured using the controlled variable method.

[0071] Specifically, control group 3 consisted of different concentrations of anti-human TCR V delta 1 Monoclonal Antibody (TS8.2), excluding anti-human TCR V delta 2 Monoclonal Antibody (15D). The remaining composition was the same as in Example 1, with the content of other components kept constant. Different concentrations of anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) were set, corresponding to concentrations of 0, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. The culture method was the same as in Example 1. The total number of cells after 14 days of culture was then detected, and the fold increase was calculated.

[0072] Specifically, control group 4 consisted of different concentrations of anti-human TCR V delta 2 Monoclonal Antibody (15D) but did not contain anti-human TCR V delta 1 Monoclonal Antibody (TS8.2). The remaining composition was the same as in Example 1, with the content of other components kept constant. Different concentrations of anti-human TCR V delta 2 Monoclonal Antibody (15D) were set, namely 0, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. The culture method was the same as in Example 1. The total number of cells after 14 days of culture was then detected, and the fold increase was calculated.

[0073] Specifically, control test group 5 consisted of combinations of different concentrations of anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and anti-human TCR V delta 2 Monoclonal Antibody (15D), with the contents of other components kept constant. The combination concentrations were 0, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. The culture method was the same as in Example 1. The total number of cells after 14 days of culture was then detected, and the fold increase was calculated.

[0074] The results of control test group 3, control test group 4, and control test group 5 are as follows: Figure 3 As shown, from Figure 3 It can be seen that the addition of anti-human TCR V delta 2 Monoclonal Antibody (15D) and / or anti-human TCR V delta 2 Monoclonal Antibody (15D) can increase the fold increase of γδT cells to a certain extent. When the combination of the two is used at a concentration of 100-200 μg / mL, the fold increase of γδT cells can be greatly increased. Therefore, as a preferred embodiment of the present invention, the recombinant antibody used is a combination of 25-200 μg / mL anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and 25-200 μg / mL anti-human TCR V delta 2 Monoclonal Antibody (15D), preferably a combination of 100 μg / mL anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and 100 μg / mL anti-human TCR V delta 2 Monoclonal Antibody (15D).

[0075] In addition, Comparative Example 1 has been added to this invention, and the details of Comparative Example 1 are as follows.

[0076] Comparative Example 1: The immune cell culture kit used consisted of components A, B, C, and D.

[0077] Component A is the basal culture medium, 2 × 1 L, including RPMI-1640;

[0078] Component B is a human recombinant antibody, 1 mL, including 100 μg / mL anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and 100 μg / mL anti-human TCR V delta 2 Monoclonal Antibody (15D);

[0079] Component C is a cell proliferation promoter, 2×1mL, 5mg / mL pamidronate disodium;

[0080] Component D consists of cytokines, 2 × 1 mL, including: 700,000 U / mL IL-2, 50 μg / mL IL-15, and 8 μg / mL IL-21.

[0081] The culture steps for γδT cells in Comparative Example 1 were the same as in Example 1.

[0082] Tests and trials

[0083] The Examples and Comparative Example 1 were tested, specifically by detecting the survival rate, purity, expansion fold, and perforin expression (a marker of cytotoxicity) of γδT cells after 14 days of culture.

[0084] Specifically, peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood using the Ficoll method (density gradient centrifugation), and the PBMC viability was detected. Then, γδT cells were cultured, and all cells were collected after 14 days of culture.

[0085] The methods for detecting cell viability are as follows:

[0086] Cells collected at different culture times were stained with AO / PI, and cell counts were performed using a cell counter to calculate cell viability and cell number, and cell expansion folds were plotted.

[0087] The cell viability results are shown in Table 1 below.

[0088] Table 1. Total cell viability of each experimental group

[0089] Group Day 0 (%) Day 14 (%) Example 1 98.53±0.51 93.00±0.78 Comparative Example 1 98.53±0.51 71.83±1.59

[0090] The methods for detecting γδT cell purity and perforin expression are as follows:

[0091] After 14 days of culture, total PBMCs were collected for γδT cell purity and perforin expression detection. Flow cytometry was used to detect γδT cell purity and perforin expression in the collected cells. The antibodies used were PerCP-Cy5.5-CD3 antibody, FITC anti-human TCRγ / δ antibody, and PE-Perforin antibody. The γδT cells were a CD3+TCRγ / δ+ cell population, and the perforin-positive population was a CD3+TCRγ / δ+Perforin+ cell population.

[0092] Depend on Figure 4 It can be seen that the γδT cells cultured using the immune cell culture kit of the present invention can be expanded by more than 200 times, which is much higher than that of Comparative Example 1, indicating that the culture kit of Example 1 can improve the expansion rate of γδT cells.

[0093] Depend on Figure 5 It can be seen that the purity of γδT cells in the total cells obtained in this invention is 82.8%, which is an increase in purity compared to Comparative Example 1. From Figure 6 It can be seen that the positive rate of γδT cell perforin expression in Example 1 is as high as 100%, which is higher than the 90% positive rate of γδT cell perforin expression in Comparative Example 1.

[0094] In summary, by comparing Example 1 and Comparative Example 1, it can be seen that adding 1-thioglycerol, taurine, β-cyclodextrin, thymidine, α-lipoic acid and β-mercaptoethanol to the cell proliferation-promoting additive in component C can ensure higher cell survival rate, proliferation rate, purity and killing activity.

[0095] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immune cell culture kit, characterized in that, The immune cell culture kit is used for culturing γδT cells and includes: basal culture medium, recombinant antibody, cytokines, and cell proliferation promoters. The basal culture medium includes RPMI-1640 medium. The cell proliferation promoters include 1-thioglycerol, taurine, β-cyclodextrin, thymidine, α-lipoic acid, β-mercaptoethanol, and bisphosphonates. The bisphosphonates are 1-10 μg / mL pamidronate disodium. The recombinant antibody is a combination of 25-200 μg / mL anti-human TCR V delta 1 monoclonal antibody (TS8.2) and 25-200 μg / mL anti-human TCR V delta 2 monoclonal antibody (15D). The cytokines include 500,000-1,000,000 U / mL IL-2, 10-100 μg / mL IL-15, and 1-10 μg / mL IL-15. IL-21, in the cell proliferation-promoting additive, wherein the concentration of 1-thioglycerol is 1-10 mg / mL, the concentration of taurine is 1-10 mg / mL, the concentration of β-cyclodextrin is 1-10 mg / mL, the concentration of thymidine is 1-10 mg / mL, the concentration of α-lipoic acid is 1-10 mg / mL, and the concentration of β-mercaptoethanol is 1-10 mM.

2. The immune cell culture kit according to claim 1, wherein the bisphosphonate is 5 μg / mL pamidronate disodium.

3. The immune cell culture kit according to claim 1, characterized in that, The recombinant antibody is a combination of 100 μg / mL anti-human TCR V delta 1 Monoclonal Antibody (TS8.2) and 100 μg / mL anti-human TCR V delta 2 Monoclonal Antibody (15D).

4. A method for in vitro culture of γδT cells, characterized in that, include: Provide an immune cell culture kit as described in claims 1-3; The culture vessel was coated with a mixture of recombinant antibody and buffer. The first mixed culture medium was obtained by mixing the basal culture medium with cytokines and cell proliferation-promoting additives. PBMCs were isolated from human peripheral blood. PBMCs were seeded into coated culture containers, and cultured in the first mixed medium for a predetermined time. During the culture process, the first mixed medium was supplemented to maintain a cell concentration of 2.0–3.5 × 10⁻⁶ cells / year. 6 cells / mL; Cells collected from the culture container after the predetermined culture time.

5. The in vitro culture method for γδT cells according to claim 4, characterized in that, It also includes adding autologous plasma to the culture container during the initial stage of culture, wherein the concentration of the autologous plasma is 1-10%, and the initial stage of culture is 0-9 days of a predetermined time.

6. The in vitro culture method for γδT cells according to claim 4, characterized in that, The first mixed culture medium is obtained by mixing basal culture medium, cytokines, and cell proliferation promoters in a ratio of 1000:1:

1.

7. The use of an immune cell culture kit as described in claims 1-3 in the preparation of γδT cells and their supernatant exosomes.

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