A culture method for improving the persistence of CAR-T cell populations

Abstract

The present invention relates to the field of cell culture technology, and in particular to a culture method for improving the persistence of a CAR-T cell group, wherein the culture method is to culture CAR-T cells with a CAR-T cell culture medium, wherein the CAR-T cell culture medium uses LONZA X-VIVO15 serum-free culture medium as a basic component, and further comprises the following components: 2 g / L cytokine microcapsules, 4 g / L fermentation complex, 20 mg / L dextran sulfate, 2 mg / mL vitamin C, 15 mM 4-hydroxyethylpiperazineethanesulfonic acid, and 1.75 mM amino acids. Each component works synergistically to provide a CAR-T cell nutrient matrix, and the obtained cell proliferation ability is stronger and the killing activity is greater. The tumor-killing cell group makes the cultured CAR-T cells have a high proliferation rate, strong tumor killing activity, improved efficacy, and effectively improved persistence.

Description

Technical Field

[0001] The present invention relates to the field of cell culture technology, and in particular to a culture method for improving the persistence of a CAR-T cell population. Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy has a very significant effect in the immunotherapy of tumors, especially for lymphocytic leukemia. At present, CAR-T cell therapy has been widely used in the treatment of hematological tumors and solid tumors. At present, CAR-T therapy has two defects: on the one hand, patients need to be pretreated with chemotherapy before CAR-T cells are reinfused, otherwise the infused CAR-T cells cannot be effectively expanded, but the chemotherapy process has great toxic side effects; on the other hand, during the in vitro culture of CAR-T, the single-chain variable fragments of CAR molecules can spontaneously aggregate independently of ligand binding. This spontaneous aggregation can cause CAR molecules to transmit continuous chronic activation signals to the cell and induce CAR-T exhaustion, apoptosis and terminal differentiation. The duration of reinfused CAR-T cells in the body is limited, which weakens the persistence and anti-tumor effect of CAR-T in the body, and many patients will relapse. Studies have shown that the higher the proportion of central memory T cells contained in CAR-T cells, the better its persistence in the body and the stronger its anti-tumor effect. Trying to reduce chronic activation signals is beneficial to the long-term maintenance of CAR-T in vivo. In CAR-T cell therapy, how to improve the persistence of CAR-T is a technical problem that needs to be solved urgently. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention proposes a culture method for improving the persistence of CAR-T cell populations.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for culturing a CAR-T cell population to improve persistence comprises the following steps:

[0006] S1: Resuspend the CAR-T cells in CAR-T cell culture medium and adjust the seeding density to 1×10 6 cells / mL, add the same number of anti-CD3 / CD28 magnetic beads as cells, mix well, and culture in a 5% CO2, 37℃ incubator;

[0007] S2: After 72 h of culture in step S1, add CAR-T cell culture medium until the cell density reaches 5×10 5 cells / mL, and continue to culture in a 5% CO2, 37°C incubator;

[0008] S3: After culture in step S2, add CAR-T cell culture medium every 3 days to maintain the cell density at 5×10 5 cells / mL.

[0009] Furthermore, the CAR-T cells are preferably CD19-CAR-T cells.

[0010] Furthermore, the CAR-T cell culture medium uses LONZA X-VIVO15 serum-free culture medium as a basic component, and the LONZA X-VIVO15 serum-free culture medium also includes the following components: cytokine microcapsules 2 g / L, fermentation complex 4 g / L, dextran sulfate 20 mg / L, vitamin C 2 mg / mL, 4-hydroxyethylpiperazineethanesulfonic acid 15 mM and amino acids 1.75 mM.

[0011] Furthermore, the amino acids are 1.2 mM L-arginine, 0.2 mM L-asparagine, 0.2 mM L-cysteine, and 0.15 mM L-glutamic acid.

[0012] Furthermore, the method for preparing the cytokine microcapsule comprises the following steps:

[0013] (1) Add polylactic acid-glycolic acid copolymer to dichloromethane and mix well to obtain a polymer solution; add IL-2 and IL-4 to deionized water and mix well to obtain an aqueous phase solution; continue to slowly add the aqueous phase solution to the polymer solution, ultrasonicate at 200-300W for 3-5 min, add to a 10 g / L polyvinyl alcohol aqueous solution, ultrasonicate at 300-400W for 3-5 min, add to a 0.5 g / L polyvinyl alcohol aqueous solution, reduce the pressure to 0.002-0.004MPa, stir at 400-500 rpm for 8-10h, centrifuge at 5000 rpm for 15-20 min, wash the precipitate with deionized water, and freeze-dry at -20°C to obtain primary microcapsules;

[0014] (2) Dissolve lignin and dopamine hydrochloride in a pH = 8, 50 mM Tris-HCl buffer, add ammonium persulfate and hydrogen peroxide, stir at 150-200 rpm for 10-12 h at room temperature, take the supernatant, dialyze it in deionized water with a 3 kD dialysis bag for 24 h, and freeze-dry to obtain lignin-polydopamine;

[0015] (3) The lignin-polydopamine obtained in step (2) is mixed with a sodium hydroxide solution with a pH value of 8, and the primary microcapsules obtained in step (1) are added, ultrasonicated at 600-800 W for 10-20 min, centrifuged at 10,000 rpm for 10-15 min, the precipitate is washed with deionized water, and freeze-dried at -20°C to obtain cytokine microcapsules.

[0016] Furthermore, in step (1), the ratio of the polylactic acid-glycolic acid copolymer to dichloromethane is 1 g:50 mL.

[0017] Furthermore, in step (1), the usage ratio of IL-2, IL-4 and deionized water is 1 mg:0.5 mg:20 mL.

[0018] Furthermore, in step (1), the volume ratio of the polymer solution to the aqueous solution is 2:1.

[0019] Furthermore, in step (1), the volume ratio of the polymer solution to the 10 g / L polyvinyl alcohol aqueous solution is 1:5.

[0020] Furthermore, in step (1), the volume ratio of the 10 g / L polyvinyl alcohol aqueous solution to the 0.5 g / L polyvinyl alcohol aqueous solution is 2:1.

[0021] Furthermore, in step (2), the ratio of lignin to buffer is 1 g:150 mL.

[0022] Furthermore, in step (2), the mass ratio of lignin to dopamine hydrochloride is 3:1.

[0023] Furthermore, in step (2), the added amounts of ammonium persulfate and hydrogen peroxide are 3 wt % and 5 wt % of the lignin mass.

[0024] Furthermore, in step (3), the ratio of the amount of lignin-polydopamine to the sodium hydroxide solution is 1 g:40 mL.

[0025] Furthermore, in step (3), the mass ratio of the primary microcapsule to the lignin-polydopamine is 1:5.

[0026] Furthermore, the method for preparing the fermentation complex comprises the following steps:

[0027] (a) Astragalus, burdock root, Spirulina maxima and chickpea were washed and dried at 60°C, crushed through an 80-mesh sieve, mixed with deionized water, and sterilized at 121°C for 15 min to obtain a fermentation substrate;

[0028] (b) The activated Bifidobacterium breve and Streptomyces cinnamomi were added at 5×10 6 CFU / mL and 1×10 6 CFU / mL was inoculated into the fermentation substrate in step (a), and cultured in a shaking incubator at 180 rpm and 30°C for 72 h, and centrifuged at 10,000 rpm for 15 min to separate the solid and liquid to obtain a precipitate and a supernatant;

[0029] (c) drying the precipitate obtained in step (b) at 60° C., adding the dried precipitate into 80 vol% ethanol solution, soaking for 40 min, ultrasonicating at 35° C. and 40 kHz for 60 min, and filtering to obtain a filtrate;

[0030] (d) The supernatant obtained in step (b) and the filtrate obtained in step (c) were mixed evenly, sterilized at 100° C. for 30 min, filtered through a 0.22 μm filter membrane, rotary evaporated at 90° C. for 2-3 h, and freeze-dried at -60° C. to obtain a fermentation complex.

[0031] Furthermore, in step (a), the amount ratio of the dried and crushed astragalus, burdock root, spirulina, chickpea and deionized water is 1 g:1 g:2 g:0.5 g:100 mL.

[0032] Furthermore, the Bifidobacterium breve and Streptomyces cinnamomi in step (b) are purchased from China General Microbiological Culture Collection Center, with numbers CGMCC 1.3001 and CGMCC 4.6972, respectively.

[0033] Furthermore, in step (c), the ratio of the dried precipitate to the ethanol solution is 1 g:10 mL.

[0034] Furthermore, in step (d), the volume ratio of the supernatant to the filtrate is 2:1.

[0035] Furthermore, the preparation of the CAR-T cell culture medium includes the following steps: mixing the components of the CAR-T cell culture medium in proportion, shaking and mixing at 4° C. for 1 h, filtering and sterilizing through a 0.22 μm filter membrane to obtain a CAR-T cell culture medium.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention prepares a fermentation complex, which can provide nutrients for CAR-T cells and support their proliferation and functional activities. The use of microorganisms to ferment plants can enhance activity and reduce toxicity. The components synergistically enhance each other and have antioxidant activity. They can maintain the metabolic stability of cells and ensure the efficiency of energy utilization. They are helpful for CAR-T cells to maintain a good growth state and functional activity during culture, and improve the persistence of CAR-T cells. They contain multiple bioactive ingredients, can increase the proliferation multiples of cells, prolong the cell culture time, maintain the expression of surface markers, and improve the tumor killing effect; they have an immunomodulatory effect, help enhance the function of CAR-T cells, improve their adaptability to the tumor microenvironment, and enhance the anti-tumor immune response; they help to improve the persistence of CAR-T cells in the body, thereby enhancing the long-term anti-tumor effect.

[0038] The present invention prepares cytokine microcapsules, and the prepared capsule wall has a relatively smooth surface, good roundness, and high mechanical strength. The cytokines IL-2 and IL-4 are encapsulated to achieve their stable and sustained release. The sustainable supply of cytokines avoids the need for frequent external additions, thereby maintaining the stability and consistency of the culture environment and reducing batch-to-batch variation. In addition, the sustained-release system of the microcapsule allows cells to grow in a mild and continuous growth factor environment, reducing cell stress caused by fluctuations in factor concentrations, thereby improving cell survival rate and health status. The present invention adopts a double emulsion-solvent volatilization method to encapsulate cytokines through polylactic acid-glycolic acid copolymer. Lignin molecules contain a large number of phenolic hydroxyl groups, which can generate free radicals under oxidative conditions to undergo free radical polymerization with dopamine. Polydopamine with different degrees of polymerization can be grafted onto lignin molecules to enhance the biological adhesion ability of lignin. The catechol structure of dopamine is introduced to enhance the affinity with cells and improve biocompatibility. It can interact with the surface of primary microcapsules through hydrogen bonding forces to promote the successful preparation of microcapsules and improve stability. Lignin-polydopamine is used as a material for microcapsules to effectively encapsulate and protect cytokines, prolong their stability in an in vitro environment, and prevent the premature release of cytokines or degradation during storage and use. At the same time, the material has high biocompatibility, can promote cell proliferation and growth, improve cell activity, and effectively improve the persistence of CAR-T cells.

[0039] The CAR-T cells of the present invention reasonably select the culture matrix, scientifically proportion, and the components work synergistically to jointly provide a nutrient matrix for the CAR-T cells, improve the proliferation ability, adjust the culture method, limit the culture environment, and optimize the culture system to obtain tumor-killing cells with stronger cell proliferation ability and greater killing activity, so that the cultured CAR-T cells have a high proliferation rate and strong tumor killing activity, improve the efficacy, and effectively improve the durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 This is a scanning electron microscope image of the cytokine microcapsule described in Example 1 of the present invention;

[0042] Figure 2 The proliferation curves of the CAR-T cell populations described in Examples 1-3 and Comparative Examples 1-2 of the present invention;

[0043] Figure 3This is the therapeutic effect of the CAR-T cells described in Examples 1-3 of the present invention and Comparative Examples 1-2. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0045] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0046] Example 1: This example provides a method for culturing a CAR-T cell population to improve persistence, comprising the following steps:

[0047] S1: Resuspend the CAR-T cells in CAR-T cell culture medium and adjust the seeding density to 1×10 6 cells / mL, add the same number of anti-CD3 / CD28 magnetic beads as cells, mix well, and culture in a 5% CO2, 37℃ incubator;

[0048] S2: After 72 h of culture in step S1, add CAR-T cell culture medium until the cell density reaches 5×10 5 cells / mL, and continue to culture in a 5% CO2, 37°C incubator;

[0049] S3: After culture in step S2, add CAR-T cell culture medium every 3 days to maintain the cell density at 5×10 5 cells / mL.

[0050] The CAR-T cell culture medium uses LONZA X-VIVO15 serum-free culture medium as a basic component, and the LONZA X-VIVO15 serum-free culture medium also includes the following components: cytokine microcapsules 2 g / L, fermentation complex 4 g / L, dextran sulfate 20 mg / L, vitamin C 2 mg / mL, 4-hydroxyethylpiperazineethanesulfonic acid 15 mM, L-arginine 1.2 mM, L-asparagine 0.2 mM, L-cysteine ​​0.2 mM and L-glutamic acid 0.15 mM.

[0051] The preparation method of the cytokine microcapsule comprises the following steps:

[0052] (1) Add polylactic acid-glycolic acid copolymer to dichloromethane and mix well to obtain a polymer solution, and the amount ratio of polylactic acid-glycolic acid copolymer to dichloromethane is 1 g:50 mL; add IL-2 and IL-4 to deionized water and mix well to obtain an aqueous phase solution, and the amount ratio of IL-2, IL-4 and deionized water is 1 mg:0.5 mg:20 mL; slowly add the aqueous phase solution to the polymer solution, and the volume ratio of the polymer solution to the aqueous phase solution is 2:1; ultrasonicate at 300W for 5 min; add to 10 g / L polyvinyl alcohol aqueous solution, and the volume ratio of the polymer solution to the 10 g / L polyvinyl alcohol aqueous solution is 1:5; ultrasonicate at 400W for 5 min; add to 0.5 g / L polyvinyl alcohol aqueous solution, and the volume ratio of the 10 g / L polyvinyl alcohol aqueous solution to the 0.5 g / L polyvinyl alcohol aqueous solution is 2:1; reduce the pressure to 0.004 MPa, stir at 500 rpm for 10 h, and centrifuge at 5000 rpm for 20 min, the precipitate was washed with deionized water and freeze-dried at -20 °C to obtain primary microcapsules;

[0053] (2) Dissolve lignin and dopamine hydrochloride in a pH = 8, 50 mM Tris-HCl buffer, add ammonium persulfate and hydrogen peroxide, the amount ratio of lignin to buffer is 1 g:150 mL, the mass ratio of lignin to dopamine hydrochloride is 3:1, the amount of ammonium persulfate and hydrogen peroxide added is 3wt% and 5wt% of the mass of lignin, stir at 200 rpm at room temperature for 12 h, take the supernatant, dialyze it in deionized water with a 3 kD dialysis bag for 24 h, and freeze-dry to obtain lignin-polydopamine;

[0054] (3) The lignin-polydopamine obtained in step (2) was mixed with a sodium hydroxide solution of pH=8, the amount ratio of lignin-polydopamine to sodium hydroxide solution was 1 g:40 mL, and the primary microcapsules obtained in step (1) were added, the mass ratio of primary microcapsules to lignin-polydopamine was 1:5, 800W ultrasound was applied for 20 min, and centrifugation was performed at 10000 rpm for 15 min. The precipitate was washed with deionized water and freeze-dried at -20°C to obtain cytokine microcapsules. The electron microscope scanning image is shown in FIG. Figure 1 As shown, it is a smooth spherical structure.

[0055] The method for preparing the fermentation complex comprises the following steps:

[0056] (a) Wash astragalus, burdock root, spirulina maxima and chickpea, dry them at 60°C, crush them through an 80-mesh sieve, add deionized water and mix them evenly; the amount ratio of the dried and crushed astragalus, burdock root, spirulina maxima, chickpea and deionized water is 1 g:1 g:2 g:0.5 g:100 mL; sterilize them at 121°C for 15 min to obtain a fermentation substrate;

[0057] (b) The activated Bifidobacterium breve and Streptomyces cinnamomi were added at 5×10 6 CFU / mL and 1×10 6 CFU / mL inoculated into the fermentation substrate of step (a), the Bifidobacterium breve and Streptomyces cinnamomi purchased from China General Microbiological Culture Collection Center, numbered CGMCC 1.3001 and CGMCC 4.6972, respectively, 180 rpm, 30 ° C shaking incubator for 72 h, 10000 rpm centrifugation for 15 min, solid-liquid separation, to obtain a precipitate and a supernatant;

[0058] (c) drying the precipitate obtained in step (b) at 60° C., adding the dried precipitate to 80 vol% ethanol solution, with the ratio of the dried precipitate to the ethanol solution being 1 g:10 mL, soaking for 40 min, ultrasonically treating at 35° C. and 40 kHz for 60 min, and filtering to obtain a filtrate;

[0059] (d) The supernatant obtained in step (b) and the filtrate obtained in step (c) were mixed uniformly at a volume ratio of the supernatant to the filtrate of 2:1, sterilized at 100° C. for 30 min, filtered through a 0.22 μm filter membrane, rotary evaporated at 90° C. for 3 h, and freeze-dried at -60° C. to obtain a fermentation complex.

[0060] The preparation of the CAR-T cell culture medium comprises the following steps: mixing the components of the CAR-T cell culture medium in proportion, shaking and mixing at 4° C. for 1 h, and filtering and sterilizing through a 0.22 μm filter membrane to obtain the CAR-T cell culture medium.

[0061] Example 2: This example provides a method for culturing a CAR-T cell population to improve persistence, comprising the following steps:

[0062] S1: Resuspend the CAR-T cells in CAR-T cell culture medium and adjust the seeding density to 1×10 6 cells / mL, add the same number of anti-CD3 / CD28 magnetic beads as cells, mix well, and culture in a 5% CO2, 37℃ incubator;

[0063] S2: After 72 h of culture in step S1, add CAR-T cell culture medium until the cell density reaches 5×10 5 cells / mL, and continue to culture in a 5% CO2, 37°C incubator;

[0064] S3: After culture in step S2, add CAR-T cell culture medium every 3 days to maintain the cell density at 5×10 5 cells / mL.

[0065] The CAR-T cell culture medium uses LONZA X-VIVO15 serum-free culture medium as a basic component, and the LONZA X-VIVO15 serum-free culture medium also includes the following components: cytokine microcapsules 2 g / L, fermentation complex 4 g / L, dextran sulfate 20 mg / L, vitamin C 2 mg / mL, 4-hydroxyethylpiperazineethanesulfonic acid 15 mM, L-arginine 1.2 mM, L-asparagine 0.2 mM, L-cysteine ​​0.2 mM and L-glutamic acid 0.15 mM.

[0066] The preparation method of the cytokine microcapsule comprises the following steps:

[0067] (1) Add polylactic acid-glycolic acid copolymer to dichloromethane and mix well to obtain a polymer solution, and the amount ratio of polylactic acid-glycolic acid copolymer to dichloromethane is 1 g:50 mL; add IL-2 and IL-4 to deionized water and mix well to obtain an aqueous phase solution, and the amount ratio of IL-2, IL-4 and deionized water is 1 mg:0.5 mg:20 mL; slowly add the aqueous phase solution to the polymer solution, and the volume ratio of the polymer solution to the aqueous phase solution is 2:1; ultrasonicate at 200W for 3 min; add to 10 g / L polyvinyl alcohol aqueous solution, and the volume ratio of the polymer solution to the 10 g / L polyvinyl alcohol aqueous solution is 1:5; ultrasonicate at 300W for 3 min; add to 0.5 g / L polyvinyl alcohol aqueous solution, and the volume ratio of the 10 g / L polyvinyl alcohol aqueous solution to the 0.5 g / L polyvinyl alcohol aqueous solution is 2:1; reduce the pressure to 0.002 MPa, stir at 400 rpm for 8 h, and then stir at 5000 rpm for 1 h. After centrifugation for 15 min, the precipitate was washed with deionized water and freeze-dried at -20 °C to obtain primary microcapsules;

[0068] (2) Dissolve lignin and dopamine hydrochloride in a pH = 8, 50 mM Tris-HCl buffer, add ammonium persulfate and hydrogen peroxide, the amount ratio of lignin to buffer is 1 g:150 mL, the mass ratio of lignin to dopamine hydrochloride is 3:1, the amount of ammonium persulfate and hydrogen peroxide added is 3wt% and 5wt% of the mass of lignin, stir at 150 rpm for 10 h at room temperature, take the supernatant, dialyze it in deionized water with a 3 kD dialysis bag for 24 h, and freeze-dry to obtain lignin-polydopamine;

[0069] (3) The lignin-polydopamine obtained in step (2) was mixed with a sodium hydroxide solution with a pH value of 8, wherein the amount ratio of lignin-polydopamine to the sodium hydroxide solution was 1 g:40 mL, and the primary microcapsules obtained in step (1) were added, wherein the mass ratio of the primary microcapsules to the lignin-polydopamine was 1:5, and the mixture was ultrasonically treated at 600 W for 10 min, centrifuged at 10,000 rpm for 10 min, and the precipitate was washed with deionized water and freeze-dried at -20°C to obtain cytokine microcapsules.

[0070] The method for preparing the fermentation complex comprises the following steps:

[0071] (a) Wash astragalus, burdock root, spirulina maxima and chickpea, dry them at 60°C, crush them through an 80-mesh sieve, add deionized water and mix them evenly; the amount ratio of the dried and crushed astragalus, burdock root, spirulina maxima, chickpea and deionized water is 1 g:1 g:2 g:0.5 g:100 mL; sterilize them at 121°C for 15 min to obtain a fermentation substrate;

[0072] (b) The activated Bifidobacterium breve and Streptomyces cinnamomi were added at 5×10 6 CFU / mL and 1×10 6 CFU / mL inoculated into the fermentation substrate of step (a), the Bifidobacterium breve and Streptomyces cinnamomi purchased from China General Microbiological Culture Collection Center, numbered CGMCC 1.3001 and CGMCC 4.6972, respectively, 180 rpm, 30 ° C shaking incubator for 72 h, 10000 rpm centrifugation for 15 min, solid-liquid separation, to obtain a precipitate and a supernatant;

[0073] (c) drying the precipitate obtained in step (b) at 60° C., adding the dried precipitate to 80 vol% ethanol solution, with the ratio of the dried precipitate to the ethanol solution being 1 g:10 mL, soaking for 40 min, ultrasonically treating at 35° C. and 40 kHz for 60 min, and filtering to obtain a filtrate;

[0074] (d) The supernatant obtained in step (b) and the filtrate obtained in step (c) were mixed uniformly at a volume ratio of the supernatant to the filtrate of 2:1, sterilized at 100° C. for 30 min, filtered through a 0.22 μm filter membrane, rotary evaporated at 90° C. for 2 h, and freeze-dried at -60° C. to obtain a fermentation complex.

[0075] The preparation of the CAR-T cell culture medium comprises the following steps: mixing the components of the CAR-T cell culture medium in proportion, shaking and mixing at 4° C. for 1 h, and filtering and sterilizing through a 0.22 μm filter membrane to obtain the CAR-T cell culture medium.

[0076] Example 3: This example provides a method for culturing a CAR-T cell population to improve persistence, comprising the following steps:

[0077] S1: Resuspend the CAR-T cells in CAR-T cell culture medium and adjust the seeding density to 1×10 6 cells / mL, add the same number of anti-CD3 / CD28 magnetic beads as cells, mix well, and culture in a 5% CO2, 37℃ incubator;

[0078] S2: After 72 h of culture in step S1, add CAR-T cell culture medium until the cell density reaches 5×10 5 cells / mL, and continue to culture in a 5% CO2, 37°C incubator;

[0079] S3: After culture in step S2, add CAR-T cell culture medium every 3 days to maintain the cell density at 5×10 5 cells / mL.

[0080] The CAR-T cell culture medium uses LONZA X-VIVO15 serum-free culture medium as a basic component, and the LONZA X-VIVO15 serum-free culture medium also includes the following components: cytokine microcapsules 2 g / L, fermentation complex 4 g / L, dextran sulfate 20 mg / L, vitamin C 2 mg / mL, 4-hydroxyethylpiperazineethanesulfonic acid 15 mM, L-arginine 1.2 mM, L-asparagine 0.2 mM, L-cysteine ​​0.2 mM and L-glutamic acid 0.15 mM.

[0081] The preparation method of the cytokine microcapsule comprises the following steps:

[0082] (1) Add polylactic acid-glycolic acid copolymer to dichloromethane and mix well to obtain a polymer solution, and the amount ratio of polylactic acid-glycolic acid copolymer to dichloromethane is 1 g:50 mL; add IL-2 and IL-4 to deionized water and mix well to obtain an aqueous phase solution, and the amount ratio of IL-2, IL-4 and deionized water is 1 mg:0.5 mg:20 mL; slowly add the aqueous phase solution to the polymer solution, and the volume ratio of the polymer solution to the aqueous phase solution is 2:1; ultrasonicate at 250W for 4 min; add to 10 g / L polyvinyl alcohol aqueous solution, and the volume ratio of the polymer solution to the 10 g / L polyvinyl alcohol aqueous solution is 1:5; ultrasonicate at 350W for 4 min; add to 0.5 g / L polyvinyl alcohol aqueous solution, and the volume ratio of the 10 g / L polyvinyl alcohol aqueous solution to the 0.5 g / L polyvinyl alcohol aqueous solution is 2:1; reduce the pressure to 0.003 MPa, stir at 450 rpm for 9 h, and then stir at 5000 rpm for 9 h. After centrifugation for 18 min, the precipitate was washed with deionized water and freeze-dried at -20 °C to obtain primary microcapsules;

[0083] (2) Dissolve lignin and dopamine hydrochloride in a pH = 8, 50 mM Tris-HCl buffer, add ammonium persulfate and hydrogen peroxide, the amount ratio of lignin to buffer is 1 g:150 mL, the mass ratio of lignin to dopamine hydrochloride is 3:1, the amount of ammonium persulfate and hydrogen peroxide added is 3wt% and 5wt% of the mass of lignin, stir at 180 rpm for 11 h at room temperature, take the supernatant, dialyze it in deionized water with a 3 kD dialysis bag for 24 h, and freeze-dry to obtain lignin-polydopamine;

[0084] (3) The lignin-polydopamine obtained in step (2) was mixed with a sodium hydroxide solution with a pH value of 8, wherein the amount ratio of lignin-polydopamine to the sodium hydroxide solution was 1 g:40 mL, and the primary microcapsules obtained in step (1) were added, wherein the mass ratio of the primary microcapsules to the lignin-polydopamine was 1:5, and the mixture was ultrasonically treated at 700 W for 15 min, centrifuged at 10,000 rpm for 12 min, and the precipitate was washed with deionized water and freeze-dried at -20°C to obtain cytokine microcapsules.

[0085] The method for preparing the fermentation complex comprises the following steps:

[0086] (a) Wash astragalus, burdock root, spirulina maxima and chickpea, dry them at 60°C, crush them through an 80-mesh sieve, add deionized water and mix them evenly; the amount ratio of the dried and crushed astragalus, burdock root, spirulina maxima, chickpea and deionized water is 1 g:1 g:2 g:0.5 g:100 mL; sterilize them at 121°C for 15 min to obtain a fermentation substrate;

[0087] (b) The activated Bifidobacterium breve and Streptomyces cinnamomi were added at 5×10 6 CFU / mL and 1×10 6 CFU / mL inoculated into the fermentation substrate of step (a), the Bifidobacterium breve and Streptomyces cinnamomi purchased from China General Microbiological Culture Collection Center, numbered CGMCC 1.3001 and CGMCC 4.6972, respectively, 180 rpm, 30 ° C shaking incubator for 72 h, 10000 rpm centrifugation for 15 min, solid-liquid separation, to obtain a precipitate and a supernatant;

[0088] (c) drying the precipitate obtained in step (b) at 60° C., adding the dried precipitate to 80 vol% ethanol solution, with the ratio of the dried precipitate to the ethanol solution being 1 g:10 mL, soaking for 40 min, ultrasonically treating at 35° C. and 40 kHz for 60 min, and filtering to obtain a filtrate;

[0089] (d) The supernatant obtained in step (b) and the filtrate obtained in step (c) were mixed uniformly at a volume ratio of the supernatant to the filtrate of 2:1, sterilized at 100° C. for 30 min, filtered through a 0.22 μm filter membrane, rotary evaporated at 90° C. for 2.5 h, and freeze-dried at -60° C. to obtain a fermentation complex.

[0090] The preparation of the CAR-T cell culture medium comprises the following steps: mixing the components of the CAR-T cell culture medium in proportion, shaking and mixing at 4° C. for 1 h, and filtering and sterilizing through a 0.22 μm filter membrane to obtain the CAR-T cell culture medium.

[0091] The only difference between Comparative Example 1 and Example 1 is that the astragalus, burdock root, Spirulina maxima and chickpea are washed and dried at 60°C, crushed and passed through an 80-mesh sieve to obtain a mixed powder, added to an 80 vol% ethanol solution, the amount ratio of the mixed powder to the ethanol solution is 1 g:10 mL, soaked for 40 min, ultrasonicated at 35°C and 40 kHz for 60 min, filtered to obtain a filtrate, the obtained filtrate is sterilized at 121°C for 30 min, filtered through a 0.22 μm filter membrane, rotary evaporated at 90°C for 2-3 h, and freeze-dried at -60°C, and the obtained product is used to replace the fermentation complex.

[0092] The difference between Comparative Example 2 and Example 1 is that cytokine microcapsules are not added, and IL-2 and IL-4 are directly added, and the added amounts are the same as the amounts of cytokines IL-2 and IL-4 provided by the cytokine microcapsules.

[0093] Experimental Example 1: On the 0th, 3rd, 5th, 7th and 9th days of culture in Example 1-3 and Comparative Example 1-2, cell counting was performed, the cell number was recorded, a cell proliferation curve was drawn, and cells cultured on the 7th and 21st days were taken for flow cytometry detection. The results are shown in FIG. Figure 2 And as shown in Table 1.

[0094] Table 1 Flow cytometry results

[0095]

[0096] Figure 2 The results in Table 1 show that the number of cells and the expression level of CD19CAR in Examples 1-3 are higher than those in Comparative Examples 1-2, indicating that the CAR-T culture medium prepared by the present invention can increase the proliferation multiple of cells and prolong the cell culture days when culturing CD19-CAR-T cells. The CAR-T cell culture method of the present invention can promote cell proliferation, maintain the expression of CD19-CAR, and improve the persistence of CAR-T cells.

[0097] Experimental Example 2: Thirty 6-week-old female NCG mice were injected with 1.2×10 6 NALM-6 Luc+ The cells were then randomly divided into 5 groups, with 6 mice in each group. One week later, each mouse in each group was injected with 1.2×10 6 The CAR-T cells of Examples 1-3 and Comparative Examples 1-2 were cultured for 9 days. Venous blood was obtained from the tail of the mice on days 22 and 28, and the CD19 expression of tumor cells in the mice was analyzed by flow cytometry. + NALM-6 Luc+ The absolute number of cancer cells. Figure 3 shown.

[0098] Figure 3 The results showed that the number of tumor cells in Example 1-3 groups was less than that in Comparative Example 1-2, and the treatment effect of Example 1-3 groups was better, indicating that the CAR-T cell culture medium of the present invention can improve the lethality of CAR-T cells and improve the persistence.

[0099] Experimental Example 3: The culture media prepared in Examples 1-3 and Comparative Examples 1-2 were used as samples to test Escherichia coli using MacConkey agar and PCR method; Staphylococcus aureus was tested using mannitol high salt agar and lectin; and Candida was tested using Sabouraud dextrose agar and Candida chromogenic medium. The experimental results are shown in Table 2.

[0100] Table 2 Bacteria test results

[0101]

[0102] It can be seen from the results in Table 2 that the CAR-T cell culture medium prepared by the present invention is safe and reliable, and has no foreign bacterial contamination.

[0103] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0104] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for culturing a CAR-T cell population to improve persistence, characterized in that: The following steps are involved: S1: Resuspend CAR-T cells in CAR-T cell culture medium and adjust the seeding density to 1×10 6 cells / mL, add the same number of anti-CD3 / CD28 magnetic beads as cells, mix well, and culture in a 5% CO2, 37℃ incubator; S2: After 72 h of culture in step S1, add CAR-T cell culture medium until the cell density reaches 5×10 5 cells / mL, and continue to culture in a 5% CO2, 37°C incubator; S3: After culture in step S2, add CAR-T cell culture medium every 3 days to maintain the cell density at 5×10 5 cells / mL; The CAR-T cells are CAR-T cells targeting CD19; The CAR-T cell culture medium uses LONZA X-VIVO15 serum-free culture medium as a basic component, and the LONZA X-VIVO15 serum-free culture medium also includes the following components: 2 g / L cytokine microcapsules, 4 g / L fermentation complex, 20 mg / L dextran sulfate, 2 mg / mL vitamin C, 15 mM 4-hydroxyethylpiperazineethanesulfonic acid, and 1.75 mM amino acids; The amino acids are L-arginine 1.2 mM, L-asparagine 0.2 mM, L-cysteine ​​0.2 mM, and L-glutamic acid 0.15 mM; The preparation method of the cytokine microcapsule comprises the following steps: (1) adding polylactic acid-co-glycolic acid copolymer to dichloromethane and mixing evenly to obtain a polymer solution; adding IL-2 and IL-4 to deionized water and mixing evenly to obtain an aqueous phase solution; continuously and slowly adding the aqueous phase solution to the polymer solution, ultrasonically adding it to a 10 g / L aqueous solution of polyvinyl alcohol, ultrasonically adding it to a 0.5 g / L aqueous solution of polyvinyl alcohol, reducing the pressure, stirring, centrifuging, washing the precipitate with deionized water, and freeze-drying to obtain primary microcapsules; (2) dissolving lignin and dopamine hydrochloride in Tris-HCl buffer, adding ammonium persulfate and hydrogen peroxide, stirring, taking the supernatant for dialysis, and freeze-drying to obtain lignin-polydopamine; (3) mixing the lignin-polydopamine obtained in step (2) with a sodium hydroxide solution, adding the primary microcapsules obtained in step (1), ultrasonicating, centrifuging, washing the precipitate with deionized water, and freeze-drying to obtain cytokine microcapsules; The method for preparing the fermentation complex comprises the following steps: (a) washing and drying astragalus, burdock root, spirulina maxima and chickpea, crushing and sieving, adding deionized water to mix, and sterilizing to obtain a fermentation substrate; (b) inoculating the activated Bifidobacterium breve and Streptomyces cinnamomi into the fermentation substrate of step (a), culturing, centrifuging, and separating the solid and liquid to obtain a precipitate and a supernatant; the Bifidobacterium breve and Streptomyces cinnamomi were purchased from the China General Microbiological Culture Collection Center, with numbers CGMCC 1.3001 and CGMCC 4.6972, respectively; (c) drying the precipitate obtained in step (b), adding the dried precipitate into an ethanol solution, soaking it, ultrasonicating it, and filtering it with suction to obtain a filtrate; (d) the supernatant obtained in step (b) and the filtrate obtained in step (c) are mixed uniformly, sterilized, filtered, rotary evaporated, and freeze-dried to obtain a fermentation complex.

2. The method for culturing a CAR-T cell population to improve persistence according to claim 1, characterized in that: In step (1), the dosage ratio of the polylactic acid-glycolic acid copolymer to dichloromethane is 1 g:50 mL; the dosage ratio of IL-2, IL-4 and deionized water is 1 mg:0.5 mg:20 mL; the volume ratio of the polymer solution to the aqueous phase solution is 2:1; the volume ratio of the polymer solution to a 10 g / L polyvinyl alcohol aqueous solution is 1:5; and the volume ratio of a 10 g / L polyvinyl alcohol aqueous solution to a 0.5 g / L polyvinyl alcohol aqueous solution is 2:

1.

3. The method for culturing a CAR-T cell population to improve persistence according to claim 1, characterized in that: In step (2), the dosage ratio of lignin to buffer solution is 1 g:150 mL; the mass ratio of lignin to dopamine hydrochloride is 3:1; and the added amounts of ammonium persulfate and hydrogen peroxide are 3wt% and 5wt% of the mass of lignin.

4. The method for culturing a CAR-T cell population to improve persistence according to claim 1, characterized in that: In step (3), the dosage ratio of the lignin-polydopamine to the sodium hydroxide solution is 1 g:40 mL; and the mass ratio of the primary microcapsule to the lignin-polydopamine is 1:

5.

5. The method for culturing CAR-T cell populations to improve persistence according to claim 1, characterized in that: The amount ratio of the dried and crushed astragalus, burdock root, spirulina maxima, chickpea and deionized water in step (a) is 1 g: 1 g: 2 g: 0.5 g: 100 mL.

6. The method for culturing CAR-T cell populations to improve persistence according to claim 1, characterized in that: The ratio of the dried precipitate to the ethanol solution in step (c) is 1 g:10 mL.

7. The method for culturing CAR-T cell populations to improve persistence according to claim 1, characterized in that: The volume ratio of the supernatant to the filtrate in step (d) is 2:1.

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