A method for expanding CAR-T cells

Abstract

The present invention discloses a method for expanding CAR-T cells, belonging to the field of biotechnology, which comprises the following steps: resuspending CAR-T cells with an expansion culture medium to obtain a cell suspension, culturing for 72 to 96 hours, and then replacing 20 to 50% of the expansion culture medium every 48 to 72 hours; the expansion culture medium comprises the following components: 3 to 7% human AB serum, 100 to 200 μg / mL IL-7, 100 to 300 μg / mL IL-15, 100 to 200 μg / mL IL-21, 2 to 10 mM N-acetylcysteine, 2 to 10 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10 to 40 μM β-mercaptoethanol, 0.01 to 0.04 mM aspartic acid, 100 to 200 ng / mL linolenic acid, 2 to 8.5 mg / mL fucoidan, 1.5 to 8 mg / mL modified chitosan, and the balance being a basal medium. By using the expansion culture medium with a specific composition for CAR-T cells, the present invention significantly improves the amplification multiple and cell viability under the combined action and mutual influence of each component, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for expanding CAR-T cells. Background Art

[0002] CAR-T cells (chimeric antigen receptor T cells) are immune cells generated by fusing T cells with antigen receptors through genetic engineering technology. They show great potential in cancer immunotherapy, especially in the treatment of hematological malignancies (such as leukemia, lymphoma, etc.) and have achieved remarkable results. CAR-T cells can recognize and specifically kill tumor cells, and their anti-tumor effect has great advantages over traditional treatment methods (such as chemotherapy, radiotherapy, etc.).

[0003] However, the effectiveness and persistence of CAR-T cells are often limited by their amplification efficiency and cell viability. To ensure the anti-tumor effect of CAR-T cells in vivo, it is crucial to expand these cells to a sufficient number and ensure that they maintain high immune function during the amplification process.

[0004] Chitosan is a natural high-molecular polysaccharide extracted from the exoskeletons of crustaceans (such as shrimps, crabs). It has a wide range of biomedical and industrial applications. The molecular structure of chitosan contains amino and hydroxyl groups, making it have excellent biocompatibility, biodegradability, and biological activity. Patent CN107129545A discloses the application of modified chitosan in the culture of CIK cells and the use of preparing commercial culture media and culture vessels. The modified chitosan prepared by a specific method can improve the amplification vitality and tumor-killing activity of CIK cells, but it does not disclose its effect in the culture of CAR-T cells.

[0005] In view of this, the present application is proposed. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for expanding CAR-T cells. The method for expanding CAR-T cells of the present invention can effectively increase the amplification multiple and improve the cell viability, and has a wide application prospect.

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

[0008] A method for expanding CAR-T cells, comprising the following steps:

[0009] Resuspend CAR-T cells with an amplification culture medium to obtain a cell suspension, culture for 72 - 96 h, and then replace 20 - 50% of the amplification culture medium every 48 - 72 h;

[0010] The amplification medium comprises the following components: 3-7% human AB serum, 100-200 μg / mL IL-7, 100-300 μg / mL IL-15, 100-200 μg / mL IL-21, 2-10 mM N-acetylcysteine, 2-10 μg / mL CD3 / CD28 monoclonal antibody-conjugated magnetic beads, 10-40 μM β-mercaptoethanol, 0.01-0.04 mM aspartic acid, 100-200 ng / mL linolenic acid, 2-8.5 mg / mL fucoidan, 1.5-8 mg / mL modified chitosan, and the balance being a basal medium.

[0011] In the present invention, the CAR-T cells are cultured in the amplification medium with the specific composition. Under the combined action and mutual influence of each component, the amplification multiple and cell viability are significantly improved, showing broad application prospects.

[0012] Among them, IL-7 (Interleukin-7): IL-7 can significantly promote the proliferation of cells during the amplification of CAR-T cells, especially in the early stage of amplification. By enhancing the cell receptor (TCR) signal and promoting the activation of proliferation signals, it helps to improve the proliferation ability of CAR-T cells. At the same time, it prevents cell exhaustion during the amplification process and reduces cell death caused by insufficient or over-activated growth factors.

[0013] IL-15 (Interleukin-15): IL-15 is a cytokine with strong immune-stimulating effects. By stimulating the IL-15 receptor on cells, it enhances cell proliferation, survival, tolerance, and anti-tumor activity, can significantly increase the amplification multiple of CAR-T cells, helps to maintain the survival state and function of cells, and reduces apoptosis.

[0014] IL-21 (Interleukin-21): IL-21 is a cytokine secreted by CD4 + T cells, mainly regulating cell proliferation, differentiation, and immune responses. IL-21 promotes cell division and proliferation by enhancing the JAK / STAT signaling pathway. By regulating cell metabolism and survival signals, IL-21 also helps to improve the tolerance of CAR-T cells and enhance cell immune activity.

[0015] In the specific medium of the present invention, by combining IL-7, IL-15, and IL-21 and strictly controlling their contents, the amplification multiple and cell viability are effectively improved.

[0016] Among them, fucoidan and modified chitosan can form a supporting structure in the culture medium, promote the adhesion and growth of CAR-T cells, facilitate the transmission of contact signals between CAR-T cells, form a stable cell attachment environment, improve cell viability, reduce the stress response of cells, help delay cell exhaustion, and to a certain extent, regulate the release effect of growth factors such as IL-7, IL-15, and IL-21, provide nutrition for cell growth, regulate the osmotic pressure during the culture process, protect the cell membrane, improve the water retention capacity of cells. At the same time, fucoidan has an antioxidant effect, and the chitosan has excellent bactericidal effects, can effectively maintain the stability of the culture environment, adsorb metabolic wastes, reduce the inhibitory effect on cell proliferation. Under the synergistic effect of the two, the cell viability and amplification multiple are effectively improved.

[0017] As a preferred embodiment of the present invention, the basal medium is X-VIVO 15 medium.

[0018] As a preferred embodiment of the present invention, the culture is carried out in an incubator at 36.5~37.5°C and 5% CO2.

[0019] As a preferred embodiment of the present invention, in the amplification medium, the mass ratio of fucoidan to modified chitosan is 1:(1~1.5).

[0020] As a preferred embodiment of the present invention, the preparation method of the modified chitosan is as follows:

[0021] Disperse chitosan and surfactant in water, stir evenly to obtain a mixed solution;

[0022] Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, adjust the pH to 5.4~5.8, react, filter, and dry to obtain modified chitosan.

[0023] As a preferred embodiment of the present invention, the surfactant is sodium dodecylbenzenesulfonate.

[0024] As a preferred embodiment of the present invention, the mass ratio of chitosan, surfactant, and water is 1:(0.05~0.12):(4~20).

[0025] As a preferred embodiment of the present invention, the mass ratio of chitosan, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(0.15~0.3):(0.15~0.3).

[0026] As a preferred embodiment of the present invention, the reaction temperature is 20~30°C and the time is 2~8h.

[0027] As a preferred embodiment of the present invention, the concentration of CAR-T cells in the cell suspension is 0.5~2×10 7 cells / mL.

[0028] The beneficial effects of the present invention are as follows: By using the amplification medium with a specific composition, the CAR-T cells of the present invention significantly improve the amplification multiple and cell viability under the combined action and mutual influence of each component, and have broad application prospects. Detailed implementation manners

[0029] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the protection scope of the present application.

[0030] In the present application, for the technical features described in an open-ended manner, a closed technical solution composed of the listed features is included, and an open technical solution including the listed features is also included.

[0031] In the present application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous, and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, each integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0032] In the present application, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0033] Unless otherwise specified, the component raw materials or instruments used in the embodiments and comparative examples of the present invention are all commercially available raw materials or instruments, and the component raw materials used in each parallel experiment are of the same kind.

[0034] Among them, the CAR-T cells of the present invention are derived from Weigen Biotech, with the number VGT-930-18C3.

[0035] The CD3 / CD28 monoclonal antibody-conjugated magnetic beads are from AAT Bioquest, product number: C-BETA2001.

[0036] Fucoidan is from Macklin Reagent, product number: F889413.

[0037] The X-VIVO 15 medium is from LONZA, product number 04-418Q.

[0038] Chitosan is sourced from Macklin Reagent, catalog number: C850347.

[0039] The following examples are provided to facilitate the understanding of the present invention. These examples are not provided to limit the scope of the claims.

[0040] Example 1

[0041] A method for expanding CAR-T cells, comprising the following steps:

[0042] Resuspend the CAR-T cells in the expansion medium and adjust the cell concentration to 1×10 7 cells / mL to obtain a cell suspension, culture it in an incubator at 37°C and 5% CO2 for 72 h, then replace 40% of the expansion medium every 48 h, and culture for a total of 10 days.

[0043] The expansion medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody-conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 4 mg / mL fucoidan, 6 mg / mL modified chitosan, and the balance X-VIVO 15 medium.

[0044] The preparation method of the modified chitosan is as follows: Disperse chitosan and sodium dodecylbenzenesulfonate in water, stir evenly at a speed of 100 rpm to obtain a mixed solution; add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, adjust the pH to 5.6, react at 26°C for 5 h, filter, and dry to obtain modified chitosan. The mass ratio of chitosan, surfactant, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.1:10:0.2:0.2.

[0045] Example 2

[0046] A method for expanding CAR-T cells, comprising the following steps:

[0047] Resuspend the CAR-T cells in the expansion medium and adjust the cell concentration to 1×10 7 cells / mL to obtain a cell suspension, culture it in an incubator at 37°C and 5% CO2 for 72 h, then replace 40% of the expansion medium every 48 h, and culture for a total of 10 days.

[0048] The amplification medium comprises the following components: 5% human AB serum, 100 μg / mL IL-7, 300 μg / mL IL-15, 100 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL anti-CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 4 mg / mL fucoidan, 6 mg / mL modified chitosan, and the balance of X-VIVO 15 medium.

[0049] The preparation method of the modified chitosan is as follows: Chitosan and sodium dodecylbenzenesulfonate are dispersed in water and stirred evenly at a speed of 100 rpm to obtain a mixed solution; 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, the pH is adjusted to 5.6, and the reaction is carried out at 26 °C for 5 h, followed by filtration and drying to obtain the modified chitosan. The mass ratio of chitosan, surfactant, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.1:10:0.2:0.2.

[0050] Example 3

[0051] A method for amplifying CAR-T cells comprises the following steps:

[0052] The CAR-T cells are resuspended with the amplification medium, and the cell concentration is adjusted to 1×10 7 cells / mL to obtain a cell suspension, which is cultured in an incubator at 37 °C and 5% CO2 for 72 h, and then 40% of the amplification medium is replaced every 48 h, and the total culture time is 10 days.

[0053] The amplification medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL anti-CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 5 mg / mL fucoidan, 5 mg / mL modified chitosan, and the balance of X-VIVO 15 medium.

[0054] The preparation method of the modified chitosan is as follows: Chitosan and sodium dodecylbenzenesulfonate are dispersed in water and stirred evenly at a speed of 100 rpm to obtain a mixed solution; 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, the pH is adjusted to 5.6, and the reaction is carried out at 26 °C for 5 h. After filtration and drying, the modified chitosan is obtained. The mass ratio of chitosan, surfactant, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.1:10:0.2:0.2.

[0055] Example 4

[0056] A method for expanding CAR-T cells includes the following steps:

[0057] Resuspend CAR-T cells with an expansion medium, adjust the cell concentration to 1×10 7 cells / mL to obtain a cell suspension, culture it in an incubator at 37 °C and 5% CO2 for 72 h, and then replace 40% of the expansion medium every 48 h for a total of 10 days.

[0058] The expansion medium includes the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody-conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 2 mg / mL fucoidan, 8 mg / mL modified chitosan, and the balance of X-VIVO 15 medium.

[0059] The preparation method of the modified chitosan is as follows: Chitosan and sodium dodecylbenzenesulfonate are dispersed in water and stirred evenly at a speed of 100 rpm to obtain a mixed solution; 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, the pH is adjusted to 5.6, and the reaction is carried out at 26 °C for 5 h. After filtration and drying, the modified chitosan is obtained. The mass ratio of chitosan, surfactant, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.1:10:0.2:0.2.

[0060] Example 5

[0061] A method for expanding CAR-T cells includes the following steps:

[0062] Resuspend CAR-T cells with an expansion medium, adjust the cell concentration to 1×10 7Cells / mL were used to obtain a cell suspension, which was cultured in an incubator at 37°C and 5% CO2 for 72 h. Thereafter, 40% of the amplification medium was replaced every 48 h, and the total culture time was 10 days.

[0063] The amplification medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody-conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 8.5 mg / mL fucoidan, 1.5 mg / mL modified chitosan, and the balance of X-VIVO 15 medium.

[0064] The preparation method of the modified chitosan is as follows: Chitosan and sodium dodecylbenzenesulfonate are dispersed in water and stirred evenly at a speed of 100 rpm to obtain a mixed solution; 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, the pH is adjusted to 5.6, and the reaction is carried out at 26°C for 5 h. Then, it is filtered and dried to obtain modified chitosan. The mass ratio of chitosan, surfactant, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:0.1:10:0.2:0.2.

[0065] Comparative Example 1

[0066] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain fucoidan, and the others are the same.

[0067] The amplification medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody-conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 6 mg / mL modified chitosan, and the balance of X-VIVO 15 medium.

[0068] Comparative Example 2

[0069] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain modified chitosan, and the others are the same.

[0070] The amplification medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 4 mg / mL fucoidan, and the balance of X-VIVO 15 medium.

[0071] Comparative Example 3

[0072] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 does not contain fucoidan (equivalent amount of modified chitosan is used to replace fucoidan), and the others are the same.

[0073] The amplification medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 10 mg / mL modified chitosan, and the balance of X-VIVO 15 medium.

[0074] Comparative Example 4

[0075] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 does not contain modified chitosan fucoidan (equivalent amount of fucoidan is used to replace modified chitosan), and the others are the same.

[0076] The amplification medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 10 mg / mL fucoidan, and the balance of X-VIVO 15 medium.

[0077] Comparative Example 5

[0078] The difference between Comparative Example 5 and Example 1 is that Comparative Example 5 uses an equivalent amount of chitosan to replace modified chitosan, and the others are the same.

[0079] The amplification medium comprises the following components: 5% human AB serum, 200 μg / mL IL-7, 100 μg / mL IL-15, 200 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 4 mg / mL fucoidan, 6 mg / mL chitosan, and the balance of X-VIVO15 medium.

[0080] Comparative Example 6

[0081] The difference between Comparative Example 6 and Example 1 is that the dosages of IL-7, IL-15, and IL-21 in Comparative Example 6 are not within the scope of the present invention (the total amount of the three is the same), and the others are the same.

[0082] The amplification medium comprises the following components: 5% human AB serum, 50 μg / mL IL-7, 400 μg / mL IL-15, 50 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 4 mg / mL fucoidan, 6 mg / mL modified chitosan, and the balance of X-VIVO15 medium.

[0083] Comparative Example 7

[0084] The difference between Comparative Example 7 and Example 1 is that the dosages of IL-7, IL-15, and IL-21 in Comparative Example 7 are not within the scope of the present invention (the total amount of the three is the same), and the others are the same.

[0085] The amplification medium comprises the following components: 5% human AB serum, 400 μg / mL IL-7, 50 μg / mL IL-15, 50 μg / mL IL-21, 5 mM N-acetylcysteine, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 20 μM β-mercaptoethanol, 0.03 mM aspartic acid, 150 ng / mL linolenic acid, 4 mg / mL fucoidan, 6 mg / mL modified chitosan, and the balance of X-VIVO15 medium.

[0086] Experimental data

[0087] Among them, the cell viability and amplification multiples of the examples and comparative examples are shown in Table 1.

[0088] Table 1

[0089]

[0090] As can be seen from Table 1, the CAR-T cell amplification method described in the present invention can effectively increase the amplification multiple and improve the cell viability, having broad application prospects.

[0091] By comparing Comparative Examples 1-2 with Comparative Examples 6-7, it can be seen that by controlling the dosages of IL-7, IL-15, and IL-21 within the scope of the present invention, the cell viability and amplification multiple are significantly improved. When the dosages of IL-7, IL-15, and IL-21 deviate from the scope of the present invention, the cell viability and amplification multiple will decrease significantly.

[0092] By comparing Comparative Example 1, 3 with Examples 4, 5, it can be seen that by controlling the mass ratio of fucoidan to modified chitosan in the amplification medium to be 1:(1-1.5), the amplification multiple and cell viability can be further improved.

[0093] By comparing Comparative Example 1 with Comparative Examples 1-5, it can be seen that the combined use of the fucoidan and the modified chitosan described in the present invention can significantly increase the amplification multiple and cell viability, and the two have a significant synergistic effect in improving the amplification multiple and cell viability under the system of the present invention.

[0094] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for expanding CAR-T cells, characterized in that, It includes the following steps: Resuspend CAR-T cells with the amplification medium to obtain a cell suspension, culture for 72 - 96 h, and then replace 20 - 50% of the amplification medium every 48 - 72 h; The amplification medium includes the following components: 3 - 7% human AB serum, 100 - 200 μg / mL IL-7, 100 - 300 μg / mL IL-15, 100 - 200 μg / mL IL-21, 2 - 10 mM N-acetylcysteine, 2 - 10 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10 - 40 μM β-mercaptoethanol, 0.01 - 0.04 mM aspartic acid, 100 - 200 ng / mL linolenic acid, 2 - 8.5 mg / mL fucoidan, 1.5 - 8 mg / mL modified chitosan, and the balance of basal medium; The concentration of CAR-T cells in the cell suspension is 0.5~2×10 7 cells / mL; The preparation method of the modified chitosan is as follows: Disperse chitosan and surfactant in water, stir evenly to obtain a mixed solution; Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, adjust the pH to 5.4 - 5.8, react, filter, and dry to obtain modified chitosan; The surfactant is sodium dodecylbenzenesulfonate.

2. The CAR-T cell expansion method according to claim 1, wherein The basal medium is X-VIVO15 medium.

3. The CAR-T cell expansion method according to claim 1, wherein, The culture is carried out in an incubator at 36.5 - 37.5 °C and 5% CO2.

4. The CAR-T cell expansion method according to claim 1, wherein In the amplification medium, the mass ratio of fucoidan to modified chitosan is 1:(1 - 1.5).

5. The CAR-T cell expansion method according to claim 1, wherein, The mass ratio of chitosan, surfactant, and water is 1:(0.05 - 0.12):(4 - 20).

6. The CAR-T cell expansion method according to claim 1, characterized in that, The mass ratio of chitosan, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:(0.15 - 0.3):(0.15 - 0.3).

7. The CAR-T cell expansion method according to claim 1, wherein The reaction temperature is 20 - 30 °C and the time is 2 - 8 h.

Images