Animal-source-free serum-free culture medium for T lymphocyte culture and application thereof

Abstract

The invention relates to the technical field of cell culture, in particular to an animal-source-free serum-free culture medium for T lymphocyte culture and application of the animal-source-free serum-free culture medium. The T lymphocyte culture medium provided by the invention does not contain animal-derived components and any substances with undefined components such as serum, any serum / blood replacement component does not need to be added when the T lymphocyte culture medium is used for culturing T lymphocytes, and compared with a traditional culture medium needing to be added with serum / blood replacement, the T lymphocyte culture medium has the advantages of small inter-batch difference and no potential pathogenic microorganism spreading risk; and higher amplification efficiency and similar T lymphocyte phenotypes can be realized compared with a culture medium added with serum / blood substitutes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to an animal-free and serum-free culture medium for T lymphocyte culture and application thereof. BACKGROUND

[0002] The nutrients required for the survival and expansion of T lymphocytes in vitro include amino acids, vitamins, sugars, lipids, inorganic salts, growth factors, etc. In order to provide these nutrients, the traditional culture medium mostly uses animal or human serum as an additive, and the commonly used ones include newborn calf serum, fetal bovine serum, human AB serum, etc. The advantage of using bovine serum is that it is abundant in supply and low in price, but the disadvantage is that it has the risk of transmitting infectious diseases and the risk of causing allergic reactions due to xenogeneic proteins, and bovine serum cannot well support the growth of human T lymphocytes. The use of human AB serum can well support the growth of human T lymphocytes, but the source of human AB serum is difficult to obtain, the supply is limited, and there is also a risk of causing contamination of immunotherapy drugs by pathogens (bacteria, fungi, mycoplasma, viruses), thereby causing the risk of spreading pathogenic microorganisms; in addition, the batch-to-batch stability of serum is difficult to control. Therefore, the culture medium with serum limits the development and application of T lymphocyte preparations.

[0003] The existing serum-free culture medium for T lymphocytes can provide the nutrients required for the survival of T cells, but cannot achieve similar culture effects as adding serum without adding autologous serum.

[0004] The continuous development of cell and gene therapy technology puts forward higher requirements for the composition and performance of the culture medium as a production material. Therefore, it is necessary to develop a serum-free, animal-free component (AOF), and does not need to add any serum / blood component to stimulate and support the rapid expansion of T lymphocytes, and the culture effect is more stable, and the culture medium with relatively stable cell phenotype is very necessary. SUMMARY

[0005] The present application provides an animal-free and serum-free culture medium for T lymphocyte culture and application thereof.

[0006] During the research and development process, this invention compared various existing serum-free culture media for T lymphocytes and found that although these media do not contain serum, they cannot achieve culture effects comparable to those with serum (including expansion efficiency, batch-to-batch variability, and T lymphocyte phenotypic stability), or simultaneously achieve all of the aforementioned culture effects. Furthermore, some serum-free T lymphocyte culture media add active ingredients from traditional Chinese medicine to improve culture effects, but the composition of these extracts is complex and unclear, leading to significant batch-to-batch variations in culture results. Other serum-free T lymphocyte culture media rely on a large number of cytokines and hormones to enhance culture effects, which inevitably increases the complexity and cost of culture medium preparation. Therefore, there is still a need to develop a serum-free T lymphocyte culture medium with a relatively simple formulation and excellent culture effects. In the process of continuously exploring the formulation of serum-free T lymphocyte culture media, this invention unexpectedly discovered that the simultaneous addition of vitamin D and putrescine can significantly improve T lymphocyte expansion efficiency, reduce batch-to-batch variability, promote T lymphocyte phenotypic stability, and enhance cell in vivo survival capacity.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] This invention provides a serum-free culture medium for T lymphocyte culture, the culture medium comprising a basal medium and a serum-free additive; wherein the serum-free additive comprises vitamin D and putrescine, and further comprises an energy matrix, complex fatty acids, complex proteins, hormones, vitamin C and inorganic salts; The serum-free culture medium contains vitamin D at a concentration of 0.8-30 mM and putrescine at a concentration of 80-500 μg / L.

[0009] This invention has found that, within the above concentration range, vitamin D and putrescine can play a synergistic role in promoting the proliferation, survival and phenotypic stability of T lymphocytes, and the culture effect of adding both at the same time is significantly improved compared with adding them separately.

[0010] The energy matrix mentioned above includes sodium pyruvate and glutamine.

[0011] The complex fatty acids include oleic acid, linoleic acid, and linolenic acid.

[0012] The complex protein includes recombinant human serum albumin, recombinant human transferrin, recombinant human insulin, and insulin-like growth factor 1 (IGF1).

[0013] The hormones mentioned include progesterone.

[0014] For the aforementioned energy matrix, complex fatty acids, complex proteins, and hormones, other components belonging to this category may be added in addition to the components listed above, provided that the culture effect of T lymphocytes is not affected.

[0015] Preferably, the energy matrix described above is composed of sodium pyruvate and glutamine.

[0016] The complex fatty acids are composed of oleic acid, linoleic acid and linolenic acid.

[0017] The complex protein is composed of recombinant human serum albumin, recombinant human transferrin, recombinant human insulin, and insulin-like growth factor 1 (IGF1).

[0018] The hormone in question is progesterone.

[0019] Through experimental verification, this invention has found that, with the addition of vitamin D and putrescine, a combination of energy matrix, complex fatty acids, complex proteins, and hormones composed solely of the above-mentioned components can achieve good in vitro culture results for T lymphocytes.

[0020] Preferably, in the serum-free culture medium, the concentration of sodium pyruvate is 3-25 mM and the concentration of glutamine is 1.5-10 mM.

[0021] In the serum-free culture medium, the concentrations of oleic acid, linoleic acid, and linolenic acid are all 8-50 μg / L.

[0022] In the serum-free culture medium, the concentrations of recombinant human serum albumin, recombinant human transferrin, recombinant human insulin, and insulin-like growth factor 1 are 80-3000 mg / L, 4-25 mg / L, 4-25 mg / L, and 0.8-5 μg / L, respectively.

[0023] In the serum-free culture medium, the concentration of progesterone is 0.8-10 nM.

[0024] Controlling the concentrations of energy matrix, complex fatty acids, complex proteins, and hormones within the aforementioned ranges is more conducive to improving the expansion efficiency and survival rate of T lymphocytes, while also helping to ensure the phenotypic stability of T lymphocytes.

[0025] The inorganic salts mentioned above preferably include selenite, manganese salts, and zinc salts.

[0026] The function of manganese and zinc salts is to provide manganese and zinc ions, so any form of manganese and zinc salt can achieve a similar effect. Examples of manganese and zinc salts include, but are not limited to, manganese sulfate, zinc sulfate, manganese chloride, and zinc chloride.

[0027] The function of selenite is to provide selenite ions; therefore, any form of selenite can achieve a similar effect. For example, the selenite may be sodium selenite, calcium selenite, etc.

[0028] Preferably, in the serum-free culture medium, the concentrations of selenite, manganese salt, and zinc salt are 0.8-30 μg / L, 4-35 μg / L, and 40-220 μg / L, respectively.

[0029] The concentration of vitamin C in the serum-free culture medium is 0.8-30 mM.

[0030] The basal culture medium mentioned above includes one or more selected from α-MEM, MEM, BME, IMDM, RPMI 1640, DMEM / F-12, DMEM / F-12K, and M199.

[0031] Preferably, the basal culture medium is RPMI 1640.

[0032] In the serum-free culture medium, the basal culture medium is used as a solvent to make up the volume of the serum-free culture medium.

[0033] As a preferred embodiment of the present invention, the serum-free culture medium uses a basal culture medium as a solvent and comprises the following components: sodium pyruvate 5-20 mM, glutamine 2-8 mM, oleic acid 10-40 μg / L, linoleic acid 10-40 μg / L, linolenic acid 10-40 μg / L, recombinant human serum albumin 100-2500 mg / L, recombinant human transferrin 5-20 mg / L, recombinant human insulin 5-20 mg / L, insulin-like growth factor 1 1-4 μg / L, vitamin C 1-25 mM, vitamin D 1-25 mM, putrescine 100-400 μg / L, sodium selenite 1-25 μg / L, progesterone 1-9 nM, manganese chloride 5-30 μg / L, and zinc sulfate 50-200 μg / L.

[0034] Preferably, the serum-free culture medium is further supplemented with IL-2.

[0035] More preferably, the concentration of IL-2 in the serum-free culture medium is 200-300 IU / mL. More preferably, it is 280-300 IU / mL.

[0036] In the serum-free culture medium, IL-2 is preferably packaged separately and added to the mixture of other components when the culture medium is used.

[0037] Preferably, the serum-free culture medium is further supplemented with antibiotics. The antibiotics are preferably penicillin and streptomycin (abbreviated as penicillin-streptomycin). The concentration of penicillin is preferably 90,000-100,000 U / L, and the concentration of streptomycin is preferably 0.09-0.1 g / L.

[0038] The serum-free culture medium provided by this invention does not contain serum or any other animal-derived components, i.e., it is free of animal-derived components. "Free of animal-derived components" means that the product does not contain any components of animal or human origin.

[0039] The present invention provides a method for preparing the serum-free culture medium described above, the method comprising the step of mixing the components.

[0040] Preferably, the method includes: dissolving each component in a solvent to prepare a concentrated mother liquor, sequentially adding the concentrated mother liquor of each component to a basal culture medium, mixing them evenly, and then adding basal culture medium to make up to the total volume.

[0041] The mixing can be achieved using a magnetic stirrer.

[0042] This invention also provides any of the following applications of the serum-free culture medium for T lymphocyte culture described above: (1) Application in T lymphocyte culture; (2) Application in the preparation of T lymphocyte preparations; (3) Application in the preparation of CAR-T cell preparations.

[0043] In (1) above, the culture is an in vitro culture.

[0044] In (2) and (3) above, the preparations include drugs, testing reagents, etc.

[0045] The present invention also provides a method for culturing T lymphocytes, the method comprising: culturing T lymphocytes or a sample containing T lymphocytes in the serum-free culture medium for T lymphocyte culture described above.

[0046] In the above method, the T lymphocytes or samples containing T lymphocytes may be derived from any one or more of blood, lymphoid tissue, bone marrow, tumor tissue, and pleural or peritoneal effusion.

[0047] The T lymphocytes or samples containing T lymphocytes can be obtained by extraction using methods such as density gradient centrifugation, immunomagnetic bead adsorption, adherence adsorption, and erythrocyte lysis.

[0048] The preferred method for culturing T lymphocytes described above includes the following steps: (1) Resuscitate T lymphocytes or samples containing T lymphocytes and then resuspend them in serum-free culture medium; (2) Inoculate the resuspension from step (1) into a cell culture plate, add CD3 / CD28 magnetic beads, and then culture at 37°C. Replace or add serum-free culture medium during the culture process. (3) Expand the T lymphocytes in the cell culture plate to a culture flask for further culture.

[0049] In this invention, the T lymphocytes are preferably human T lymphocytes.

[0050] The beneficial effects of the present invention include at least the following: the T lymphocyte culture medium provided by the present invention contains no animal-derived components, does not contain any substances with unclear components such as serum, and does not require the addition of serum / blood substitutes when culturing T lymphocytes. Compared with traditional culture media that require the addition of serum / blood substitutes, it has the advantages of small batch-to-batch variability and no potential risk of pathogenic microorganism transmission. It can support the large-scale expansion of T lymphocytes with high expansion efficiency, support low-density seeding and efficient expansion culture of T lymphocytes, ensure the stability of T lymphocyte phenotype, and achieve a T lymphocyte phenotype similar to that of culture media with added serum / blood substitutes and better expansion efficiency. At the same time, it ensures a high Tcm ratio, which is conducive to achieving better cell in vivo survival capacity. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 The results show the T lymphocyte amplification efficiency detection in the experimental examples of this invention.

[0053] Figure 2 The results of T lymphocyte viability detection in the experimental examples of this invention are shown.

[0054] Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 The CD8+ cells in T lymphocytes cultured using the serum-free culture media of Examples 1-3, Comparative Examples 1-2, serum-added culture media, and serum-added culture media in the experimental examples of this invention are respectively: CD8+ cells in T lymphocytes cultured using the serum-free culture media of Examples 1-3, Comparative Examples 1-2, serum-added culture media, and serum-added culture media. + / CD4 + The results of the percentage test.

[0055] Figure 10 This is the result of detecting the proportion of central memory cells in the T lymphocyte population obtained in the experimental examples of this invention. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0057] Example 1

[0058] This embodiment provides a serum-free culture medium for T lymphocyte culture, the composition of which is as follows: sodium pyruvate 5mM, glutamine 2mM, oleic acid 10μg / L, linoleic acid 10μg / L, linolenic acid 10μg / L, recombinant human serum albumin 100mg / L, recombinant human transferrin 5mg / L, recombinant human insulin 5mg / L, IGF1 1μg / L, vitamin C 1mM, vitamin D 1mM, putrescine 100μg / L, sodium selenite 1μg / L, progesterone 1nM, manganese chloride 5μg / L, zinc sulfate 50μg / L, and RPMI 1640 to make up to 1L.

[0059] When using the serum-free culture medium, add IL-2 to a final concentration of 300 IU / mL and 1× penicillin-streptomycin (i.e., the final concentration of penicillin is 100,000 U / L and the final concentration of streptomycin is 0.1 g / L).

[0060] Example 2

[0061] This embodiment provides a serum-free culture medium for T lymphocyte culture, the composition of which is as follows: sodium pyruvate 10mM, glutamine 4mM, oleic acid 20μg / L, linoleic acid 20μg / L, linolenic acid 20μg / L, recombinant human serum albumin 500mg / L, recombinant human transferrin 10mg / L, recombinant human insulin 10mg / L, IGF1 2μg / L, vitamin C 5mM, vitamin D 5mM, putrescine 200μg / L, sodium selenite 5μg / L, progesterone 3nM, manganese chloride 15μg / L, zinc sulfate 100μg / L, and RPMI 1640 to make up to 1L.

[0062] When using the serum-free culture medium, add IL-2 to a final concentration of 300 IU / mL and 1× penicillin-streptomycin (i.e., the final concentration of penicillin is 100,000 U / L and the final concentration of streptomycin is 0.1 g / L).

[0063] Example 3

[0064] This embodiment provides a serum-free culture medium for T lymphocyte culture, the composition of which is as follows: sodium pyruvate 20mM, glutamine 8mM, oleic acid 40μg / L, linoleic acid 40μg / L, linolenic acid 40μg / L, recombinant human serum albumin 2500mg / L, recombinant human transferrin 20mg / L, recombinant human insulin 20mg / L, IGF1 4μg / L, vitamin C 25mM, vitamin D 25mM, putrescine 400μg / L, sodium selenite 25μg / L, progesterone 9nM, manganese chloride 30μg / L, zinc sulfate 200μg / L, and RPMI 1640 to make up to 1L.

[0065] When using the serum-free culture medium, add IL-2 to a final concentration of 300 IU / mL and 1× penicillin-streptomycin (i.e., the final concentration of penicillin is 100,000 U / L and the final concentration of streptomycin is 0.1 g / L).

[0066] Example 4

[0067] This embodiment provides a method for culturing T lymphocytes, which involves culturing T lymphocytes using the serum-free culture medium for T lymphocyte culture described in Examples 1, 2, or 3, and includes the following steps: (1) Add 200-300 IU / mL of IL-2 and 1× penicillin to the serum-free culture medium for T lymphocyte culture to prepare a complete culture medium; (2) Resuscitate peripheral blood mononuclear cells (PBMCs) in a 37°C water bath, centrifuge and resuspend PBMCs in the complete culture medium of step (1); (3) The PBMCs resuspended in step (2) were seeded into a six-well plate at a ratio of 3E6 per well. CD3 / CD28 magnetic beads were added to the well plate at a ratio of magnetic beads:PBMC = 1.5:1. The six-well plate was placed in a CO2 incubator at 37°C for incubation. (4) On the 5th day of culture, cell counts were performed. Based on the count, the cell density was adjusted to 5e5 cells / mL of complete culture medium. On the same day, the cells in the well plate were expanded to T25 culture flasks for further culture. Subsequently, the cells were counted and adjusted to the above density every 2-3 days for passage. (5) Harvest cells after 11 days of culture.

[0068] Comparative Example 1

[0069] This comparative example provides a serum-free culture medium for T lymphocyte culture, the composition of which differs from the serum-free culture medium of Example 1 only in that 1 mM vitamin D is removed.

[0070] Comparative Example 2

[0071] This comparative example provides a serum-free culture medium for T lymphocyte culture, the composition of which differs from the serum-free culture medium of Example 1 only in that 100 μg / L putrescine is removed.

[0072] Experimental Example

[0073] T lymphocytes were cultured using serum-free culture medium and conventional culture medium plus AB serum or blood substitute, respectively, according to the method of Example 4. The groupings were as follows: 1: Serum-free culture medium for T lymphocytes cultured in Example 1 (with added IL-2 and penicillin-streptomycin); 2: Serum-free culture medium for T lymphocytes cultured in Example 2 (with added IL-2 and penicillin-streptomycin); 3: Serum-free culture medium for T lymphocytes cultured in Example 3 (with added IL-2 and penicillin-streptomycin); 4: Serum-free culture medium for T lymphocytes cultured in Comparative Example 1 (with added IL-2 and penicillin-streptomycin); 5: Serum-free culture medium for T lymphocytes cultured in Comparative Example 2 (with added IL-2 and penicillin-streptomycin); 6: RPMI 1640 medium + AB serum (Gemini); 7: RPMI 1640 medium + blood substitute (Biolegend).

[0074] The testing methods and results are as follows.

[0075] 1. Cell expansion efficiency detection

[0076] PBMCs from three different donors were cultured using CD3 / CD28 magnetic beads to activate them. Cell counts were performed on days 6, 8, and 11 to calculate the proliferation rate.

[0077] The results are as follows Figure 1 As shown in the figure, the results represent the average proliferation rate of T lymphocytes after culture from three different donor PMBCs. The results indicate that the serum-free culture medium used in Examples 1-3 of this invention exhibits a significantly higher average proliferation rate of T lymphocytes compared to Comparative Examples 1-2 and the culture medium supplemented with serum, and the proliferation rate is more stable among different donors. In particular, the proliferation rate achieved by the serum-free culture medium in Example 1 of this invention is far higher than that of Comparative Examples 1 and 2, which use putrescine and vitamin D alone. Therefore, putrescine and vitamin D in the serum-free culture medium of this invention have a synergistic effect in promoting T lymphocyte proliferation.

[0078] 2. Cell viability detection

[0079] PBMCs were activated using CD3 / CD28 magnetic beads, and PBMCs from three different donors were cultured. Cell counts were performed on days 6, 8, and 11, and cell viability was detected by trypan blue staining.

[0080] The results are as followsFigure 2 As shown in the figure, the results represent the average T lymphocyte viability after culture from three different donor PMBCs. The results indicate that the T lymphocyte viability cultured using the serum-free culture medium of Examples 1-3 of this invention is more stable among different donors than the serum-free culture medium of Comparative Examples 1-2 and the culture medium supplemented with blood substitute / serum.

[0081] 3. CD8 + / CD4 + Percentage detection

[0082] PBMCs were activated using CD3 / CD28 magnetic beads, and PBMCs from three different donors were cultured. T cell flow cytometry phenotyping was performed on days 0, 4, 8, and 11.

[0083] The results are as follows Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 As shown, CD4+ T lymphocytes cultured using the serum-free culture medium of Examples 1-3 of the present invention and the serum-free culture medium and culture medium supplemented with blood substitute / serum of Comparative Examples 1-2 are compared. + / CD8 + The trends were similar, both showing a gradual increase in the proportion of cells with the CD8 phenotype and a gradual decrease in the proportion of cells with the CD4 phenotype.

[0084] 4. Central Memory Cell Population (Tcm) Detection

[0085] PBMCs from three different donors were cultured using CD3 / CD28 magnetic beads for activation. T cell flow cytometry phenotyping analysis compared the effects of different culture media on CD3 activation at approximately 100-fold expansion. + The influence of Tcm percentage in cells.

[0086] The results are as follows Figure 10 As shown, the Tcm percentage of T lymphocytes cultured using the serum-free culture medium of Examples 1-3 of the present invention is higher than that of the serum-free culture medium of Comparative Examples 1-2. A higher Tcm percentage means better cell survival ability. The Tcm percentage of T lymphocytes cultured using the serum-free culture medium of Examples 1-3 of the present invention is similar to that of culture medium with added blood substitute / serum.

[0087] 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; 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; and these 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. A serum-free medium for culturing T lymphocytes, characterized by comprising The culture medium comprises a basic medium and a serum-free additive; wherein the serum-free additive comprises vitamin D and putrescine, and further comprises an energy substrate, a complex fatty acid, a complex protein, a hormone, vitamin C and an inorganic salt; In the serum-free culture medium, the concentration of vitamin D is 0.8-30 mM, and the concentration of putrescine is 80-500 μg / L.

2. The serum-free culture medium for T lymphocyte culture according to claim 1, characterized by, The energy substrate comprises sodium pyruvate and glutamine; And / or, the complex fatty acid comprises oleic acid, linoleic acid and linolenic acid; And / or, the complex protein comprises recombinant human blood albumin, recombinant human transferrin, recombinant human insulin and insulin-like growth factor 1; And / or, the hormone comprises progesterone.

3. The serum-free culture medium for T lymphocyte culture according to claim 2, characterized by, In the serum-free culture medium, the concentration of sodium pyruvate is 3-25 mM, and the concentration of glutamine is 1.5-10 mM; And / or, the concentrations of oleic acid, linoleic acid and linolenic acid are all 8-50 μg / L; And / or, the concentrations of recombinant human blood albumin, recombinant human transferrin, recombinant human insulin and insulin-like growth factor 1 are respectively: 80-3000 mg / L, 4-25 mg / L, 4-25 mg / L and 0.8-5 μg / L; And / or, the concentration of progesterone is 0.8-10 nM.

4. The serum-free culture medium for T lymphocyte culture according to claim 1, characterized by, The inorganic salt comprises selenite, manganese salt and zinc salt.

5. The serum-free culture medium for T lymphocyte culture according to claim 4, characterized by In the serum-free culture medium, the concentrations of selenite, manganese salt and zinc salt are respectively: 0.8-30 μg / L, 4-35 μg / L and 40-220 μg / L; And / or, the concentration of vitamin C is 0.8-30 mM.

6. The serum-free culture medium for T lymphocyte culture according to claim 1, characterized by, The basic medium comprises one or more selected from α-MEM, MEM, BME, IMDM, RPMI 1640, DMEM / F-12, DMEM / F-12K and M199.

7. The serum-free culture medium for T lymphocyte culture according to any one of claims 1 to 6, characterized in that, The serum-free culture medium comprises the following components in the basic medium as a solvent: sodium pyruvate 5-20 mM, glutamine 2-8 mM, oleic acid 10-40 μg / L, linoleic acid 10-40 μg / L, linolenic acid 10-40 μg / L, recombinant human blood albumin 100-2500 mg / L, recombinant human transferrin 5-20 mg / L, recombinant human insulin 5-20 mg / L, insulin-like growth factor 1 1-4 μg / L, vitamin C 1-25 mM, vitamin D 1-25 mM, putrescine 100-400 μg / L, sodium selenite 1-25 μg / L, progesterone 1-9 nM, manganese chloride 5-30 μg / L, zinc sulfate 50-200 μg / L.

8. The serum-free culture medium for T lymphocyte culture according to claim 7, characterized by, The serum-free culture medium is further added with IL-2.

9. The serum-free culture medium for T lymphocyte culture according to any one of claims 1-8 is used in the following applications: (1) in T lymphocyte culture; (2) in preparation of a T lymphocyte preparation; (3) in preparation of a CAR-T cell preparation.

10. A method for culturing T lymphocytes, characterized by, The method comprises culturing T lymphocytes or a sample containing T lymphocytes in the serum-free culture medium for T lymphocyte culture according to any one of claims 1-8.

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