Preparation method and application of tumor infiltration lymphocyte

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of tumor infiltrating lymphocytes. Keratinase modified by myristoyl coenzyme A cooperates with fibronectin and elastase, so that tumor cells quickly fall off from tumor blocks, and loose tissues are subjected to low-temperature enzymolysis by protease-K, so that the phenotype and activity of tumor infiltrating lymphocytes can be protected; an eicosapentaenoic acid solution is mixed with polysorbate, ultrapure water and a culture medium matrix to obtain an emulsion mixed solution, the emulsion mixed solution is subjected to rotary evaporation to remove a solvent, and the amplification-promoting nanoparticles obtained after freeze drying can make tumor infiltrating lymphocytes ingest effective components more quickly; the tumor infiltrating lymphocytes extracted by the invention are high in activity and rapid in proliferation and growth, still have high cell activity after cryopreservation, and have good anti-breast tumor activity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically refers to a preparation method of tumor infiltrating lymphocytes and application thereof. Background Art

[0002] Tumor infiltrating lymphocytes are a type of heterogeneous lymphocytes present in the tumor microenvironment, and they play an important role in tumor immune response. Tumor infiltrating lymphocytes mainly include cytotoxic T cells, helper T cells, regulatory T cells and B cells, etc. They can directly or indirectly participate in the immune response and affect tumor growth and treatment response. The role of tumor infiltrating lymphocytes is mainly reflected in two aspects: on the one hand, they can identify and kill tumor cells and exert anti-tumor effects; on the other hand, some tumor infiltrating lymphocytes in the tumor environment, especially regulatory T cells, can inhibit the immune response and promote tumor immune escape.

[0003] Lymphocytes in the tumor microenvironment have the natural ability to recognize and attack cancer cells. By extracting, amplifying and re-injecting these cells, the patient's immune response can be greatly enhanced, thereby attacking and eliminating tumor cells. The therapeutic mechanism of tumor-infiltrating lymphocyte therapy is based on a deep understanding of the tumor microenvironment and the precise manipulation of immune cells. After years of basic research and clinical trials, this therapy has shown significant efficacy against a variety of solid tumors, especially refractory cancers such as melanoma.

[0004] At present, the existing preparation technology of tumor infiltrating lymphocytes has the following problems: First, in the preparation process of existing tumor infiltrating lymphocytes, the cell activity of the prepared tumor infiltrating lymphocytes is reduced during the enzymatic digestion process, affecting the integrity and function of the cells; Second, in the preparation of existing tumor infiltrating lymphocytes, the prepared tumor infiltrating lymphocytes have low amplification efficiency and a complicated preparation process; Third, in the preparation of existing tumor infiltrating lymphocytes, the activity retention and proliferation ability of the prepared tumor infiltrating lymphocytes are affected during the storage process. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method for preparing tumor infiltrating lymphocytes and an application thereof. In order to solve the problems that the existing preparation technology of tumor infiltrating lymphocytes affects the integrity and function of the cells, the amplification efficiency of the prepared tumor infiltrating lymphocytes is low, and the proliferation ability is affected during the storage process, the present invention uses modified keratinase for enzymatic hydrolysis and then inoculates it into a rapid amplification culture medium, and uses a stable preservation solution for cryopreservation. The prepared tumor infiltrating lymphocytes have high activity, rapid proliferation and growth, and still have high cell activity after cryopreservation.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: the present invention proposes a method for preparing tumor infiltrating lymphocytes, which specifically comprises the following steps: Step 1: Place fresh tumor tissue in a 10 cm culture medium, wash with PBS buffer 2-3 times, use a sterile scalpel to remove the necrotic area and connective tissue on the fresh tumor tissue, and cut into 1-3 mm 3 of tumor mass; Step 2, placing a keratinase solution with a mass concentration of 1.2-1.4%, a myristoyl-CoA solution of 0.4-0.7%, and an N-myristoyl transferase solution of 1.3-1.5% in a reactor with a power of 2.2-2.4 kW, the reaction temperature is 20-37°C, the reaction time is 3.2-3.6 h, the reaction speed is 200 r / min, and after the reaction, it is placed in a freeze spray dryer with a power of 2.3-2.8 kW, the freeze spray drying temperature is -30°C, the freeze spray drying particle size is 250 mm, and the freeze spray drying time is 1.3-1.4 h to obtain a modified keratinase; Step 3: Place the modified keratinase prepared in step 2, fibronectin, elastase, and RPMI-1640 culture medium in a magnetic stirrer with a power of 1.2-1.4 kW, stir at a speed of 300-350 r / min, at a temperature of 20-25° C., for 10-15 min, and stir to mix to obtain a preliminary enzymatic solution; Step 4: Put the preliminary enzymatic hydrolysate prepared in step 3 and the tumor block prepared in step 1 into a centrifuge tube, and place it in a water bath with a power of 2.2 kW, a water bath temperature of 37° C., a water bath time of 0.8-1.3 h, and perform enzymatic hydrolysis in a water bath to obtain loose tissue; Step 5: Rinse the loose tissue prepared in step 4 with 0.9% sterile saline for 2-3 times, add proteinase-K and RPMI-1640 culture medium, put into a centrifuge tube, and place in a water bath with a power of 2.2 kW. The water bath temperature is 20°C and the water bath time is 1-2 h. After low-temperature enzymolysis, filter with a 200-mesh stainless steel filter to obtain a cell suspension; Step 6: Place the cell suspension prepared in step 5 into a centrifuge with a power of 2.6-2.9 kW, centrifuge at a speed of 12000 r / min, a centrifuge temperature of 2°C, a centrifuge time of 10-15 min, discard the supernatant, and obtain precipitated cells; Step 7: After resuspending the precipitated cells prepared in step 6 with RPMI-1640 medium, mix with lymphocyte separation solution using a 25 mL pipette, put into a centrifuge with a power of 2.6-2.9 kW, the centrifugal speed is 12000 r / min, the centrifugal temperature is 2°C, the centrifugal time is 10-15 min, and after centrifugation, use a 10 mL pipette to absorb the upper cell layer to obtain a suspension of tumor-infiltrating lymphocytes after separation; Step 8: Inoculate the isolated tumor-infiltrating lymphocyte suspension prepared in step 7 into a rapid expansion medium, place in a CO2 constant temperature and humidity incubator, and culture at a constant temperature of 37°C and a CO2 volume fraction of 5%. Change the medium 1-2 times a week, and culture for 14-18 days to obtain a tumor-infiltrating lymphocyte culture medium. Step 9: Place the tumor infiltrating lymphocyte culture medium prepared in step 8 into a centrifuge with a power of 2.6-2.9 kW, a centrifugal speed of 12000 r / min, a centrifugal temperature of 2°C, a centrifugal time of 10-15 min, and after centrifugation, discard the supernatant to obtain tumor infiltrating lymphocytes.

[0007] Furthermore, in step one, the fresh tumor tissue is derived from liver cancer tumor tissue, lung cancer tumor tissue, or gastric cancer tumor tissue, and the pH of the PBS buffer is 7.4.

[0008] Furthermore, in step 2, the volume ratio of the keratinase solution, myristoyl-CoA solution, and N-myristoyltransferase solution is 1-3:0.8-1.2:1.

[0009] Furthermore, in step three, the weight ratio of the modified keratinase, fibronectin and elastase is 4-6:2-3:1, and the mass fraction of the elastase in the RPMI-1640 culture medium is 0.4-0.9%.

[0010] Furthermore, in step five, the mass fraction of the proteinase-K in the RPMI-1640 culture medium is 1.7-2.1%.

[0011] Preferably, in step eight, the method for preparing the rapid expansion medium specifically comprises the following steps: S1. Put X-VIVO 15 serum-free medium, human AB serum, cytokine composition, insulin, Lentinan, and sodium pyruvate into a stirrer with a power of 1.2-1.6 kW, stir at a temperature of 20-28° C., stir for 18-26 min, and stir at a speed of 300-500 r / min to mix and obtain a culture medium matrix; S2. Place eicosapentaenoic acid and anhydrous ethanol in a stirrer with a power of 1.2-1.6 kW, stir at a temperature of 25-28° C., stir for 13-18 min, and stir at a speed of 300-400 r / min to fully dissolve to obtain an eicosapentaenoic acid solution; S3, adding polysorbate, ultrapure water, and the culture medium matrix prepared in S1 into the eicosapentaenoic acid solution prepared in S2, stirring at a temperature of 40°C, a stirring time of 25-30 min, and a stirring speed of 400-600 r / min, stirring and mixing to obtain an emulsion mixture; S4, placing the emulsion mixture prepared in S3 into a rotary evaporator with a power of 2.5 kW, the rotary evaporation temperature is 30-35°C, the rotary evaporation time is 1.5-2 h, the rotary evaporation speed is 120-150 r / min, and then placing it into a freeze dryer with a power of 0.8-1.2 kW, the freeze drying temperature is 4°C, the freeze drying time is 12-16 h, and freeze drying is performed to obtain amplification-promoting nanoparticles; S5. Place the amplification-promoting nanoparticles prepared by S4 into the culture medium matrix prepared by S1, and place it in an ultrasonic agitator with a power of 1.8 kW, an ultrasonic frequency of 15 kHz, a stirring speed of 20,000 r / min, a stirring temperature of 25°C, and a stirring time of 10 min to obtain a rapid amplification culture medium.

[0012] Further, in S1, the mass fraction of the human AB serum in the culture medium matrix is ​​10%, the volume ratio of the human AB serum to the X-VIVO 15 serum-free culture medium is 1:9, the mass fraction of the cytokine combination in the culture medium matrix is ​​8-10%, including: 300 U / mL IL-2, 300 U / mL IL-15, 300 U / mL IL-21, 200 pg / mL epidermal growth factor, 30 ng / mL CD3 antibody, 30 ng / mL CD28 antibody and 20 ng / mL LAG-3 antibody, the mass fraction of the insulin in the culture medium matrix is ​​2.5-3.8%, and the weight ratio of the lentinan, sodium pyruvate and insulin is 2-3:1-3:1.

[0013] Furthermore, in S2, the mass fraction of eicosapentaenoic acid in anhydrous ethanol is 34-37%.

[0014] Furthermore, in S3, the mass fraction of the polysorbate in ultrapure water is 28-36%.

[0015] The present invention also proposes a method for preparing tumor infiltrating lymphocytes and the use of the prepared tumor infiltrating lymphocytes in a drug for treating breast cancer, which specifically comprises the following steps: I. Thawing the preserved tumor-infiltrating lymphocytes at 37°C, placing them in a centrifuge with a power of 2.6-2.9 kW, a centrifugal speed of 12000 r / min, a centrifugal temperature of 2°C, and a centrifugal time of 10-15 min. After centrifugation, removing the lower precipitate to obtain the tumor-infiltrating lymphocytes; II. Mixing the tumor infiltrating lymphocytes obtained in step I with pharmaceutically acceptable excipients to obtain a drug for treating breast cancer.

[0016] Furthermore, in step II, the pharmaceutically acceptable excipients include a combination of at least two of solvents, co-solvents, antibacterial agents, energy mixtures, antioxidants, anticoagulants, cell protectants, and stabilizers.

[0017] The beneficial effects achieved by the present invention are as follows: The invention adopts a mixture of myristoyl coenzyme A, N-myristoyl transferase and keratinase to react. The keratinase can specifically identify and enzymatically degrade enzymatic keratin on the surface of tumor cell membranes. Under the catalytic action of N-myristoyl transferase, myristic acid is transferred from myristoyl coenzyme A to the amino group of the N-terminal glycine residue of the protein molecule in the keratinase to form an amide bond, thereby increasing the hydrophobicity of the keratinase and more efficiently anchoring the keratinase to the tumor cell membrane. After modification, the keratinase synergizes with fibronectin and elastase capable of enzymatically degrading the extracellular matrix, so that the tumor cells quickly fall off the tumor mass. The loose tissues removed from the tumor cells are mixed with proteinase-K for enzymatic decomposition under low temperature conditions, thereby protecting the tumor. The invention discloses a novel nanoparticle preparation method for treating tumor infiltrating lymphocytes. The invention discloses a nanoparticle preparation method for treating tumor infiltrating lymphocytes. The nanoparticles are used to prepare a nanoparticle preparation ... BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the present invention or the technical solutions in the prior art, the following will be described in a clear and understandable manner in conjunction with the accompanying drawings. Obviously, the drawings described below are only for the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1This is a microscope observation image of the activated tumor-infiltrating lymphocytes prepared in Example 1 of the present invention on the 4th day of culture (400x); Figure 2 This is a result diagram of the proliferation rate of tumor infiltrating lymphocytes described in Experimental Example 1 of the present invention; Figure 3 This is a graph showing the purity of tumor infiltrating lymphocytes described in Experimental Example 1 of the present invention; Figure 4 This is a result diagram of the breast cancer tumor inhibition rate described in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0022] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials and test strains used in the following examples are purchased from commercial channels unless otherwise specified.

[0023] Example 1: This example provides a method for preparing tumor infiltrating lymphocytes, which specifically includes the following steps: Step 1: Place fresh liver cancer tumor tissue in a 10 cm culture medium, wash twice with PBS buffer, remove the necrotic area and connective tissue on the fresh tumor tissue with a sterile scalpel, and cut into 1 mm 3 of tumor mass; Step 2: Put a keratinase solution with a mass concentration of 1.2%, a 0.4% myristoyl-CoA solution, and a 1.3% N-myristoyl transferase solution into a reactor with a power of 2.2 kW, the reaction temperature is 20°C, the reaction time is 3.2 h, the reaction speed is 200 r / min, and after the reaction, put it into a freeze-spray dryer with a power of 2.3 kW, the freeze-spray drying temperature is -30°C, the freeze-spray drying particle size is 250 mm, and the freeze-spray drying time is 1.3 h to obtain a modified keratinase; Step 3: Place the modified keratinase prepared in step 2, fibronectin, elastase, and RPMI 1640 culture medium in a magnetic stirrer with a power of 1.2 kW, stir at a speed of 300 r / min, a temperature of 20°C, and a stirring time of 10 min, stir and mix to obtain a preliminary enzymatic solution; Step 4: Put the preliminary enzymatic hydrolysate prepared in step 3 and the tumor block prepared in step 1 into a centrifuge tube, and place it in a water bath with a power of 2.2 kW, a water bath temperature of 37° C., a water bath time of 0.8 h, and perform enzymatic hydrolysis in a water bath to obtain loose tissue; Step 5: Rinse the loose tissue prepared in step 4 twice with 0.9% sterile saline, add proteinase-K and RPMI 1640 culture medium, put into a centrifuge tube, and place in a water bath with a power of 2.2 kW. The water bath temperature is 20°C and the water bath time is 1 h. After low-temperature enzymolysis, filter with a 200-mesh stainless steel filter to obtain a cell suspension; Step 6: Place the cell suspension prepared in step 5 into a centrifuge with a power of 2.6 kW, centrifuge at a speed of 12000 r / min, a centrifuge temperature of 2°C, and a centrifuge time of 10 min, discard the supernatant, and obtain the precipitated cells; Step 7: After resuspending the precipitated cells prepared in step 6 with RPMI-1640 medium, mix with lymphocyte separation solution using a 25 mL pipette, put into a centrifuge with a power of 2.6 kW, the centrifugal speed is 12000 r / min, the centrifugal temperature is 2°C, the centrifugal time is 10 min, and after centrifugation, use a 10 mL pipette to absorb the upper cell layer to obtain a suspension of tumor-infiltrating lymphocytes after separation; Step 8: Inoculate the isolated tumor-infiltrating lymphocyte suspension prepared in step 7 into a rapid expansion medium, place in a CO2 constant temperature and humidity incubator, and culture at a constant temperature of 37°C and a CO2 volume fraction of 5%. Change the medium once a week and culture for 14 days to obtain a tumor-infiltrating lymphocyte culture medium. Step 9: Place the tumor-infiltrating lymphocyte culture medium prepared in step 8 into a centrifuge with a power of 2.6 kW, a centrifugal speed of 12000 r / min, a centrifugal temperature of 2°C, and a centrifugal time of 10 min. After centrifugation, discard the supernatant to obtain tumor-infiltrating lymphocytes.

[0024] In step 1, the pH of the PBS buffer is 7.4.

[0025] In step 2, the volume ratio of the keratinase solution, myristoyl-CoA solution and N-myristoyl transferase solution is 1:0.8:1.

[0026] In step three, the weight ratio of the modified keratinase, fibronectin and elastase is 4:2:1, and the mass fraction of the elastase in the RPMI-1640 culture medium is 0.4%.

[0027] In step 5, the mass fraction of the proteinase-K in the RPMI-1640 medium is 1.7%.

[0028] In step eight, the method for preparing the rapid expansion medium specifically comprises the following steps: S1. Put X-VIVO 15 serum-free medium, human AB serum, cytokine composition, insulin, Lentinan, and sodium pyruvate into a stirrer with a power of 1.2 kW, stir at a temperature of 20° C., stir for 18 min, and stir at a speed of 300 r / min to mix and obtain a culture medium matrix; S2, placing eicosapentaenoic acid and anhydrous ethanol in a 1.2 kW stirrer at a stirring temperature of 25°C, a stirring time of 13 min, and a stirring speed of 300 r / min, until the eicosapentaenoic acid solution is fully dissolved; S3, adding polysorbate, ultrapure water, and the culture medium matrix prepared in S1 into the eicosapentaenoic acid solution prepared in S2, stirring at a temperature of 40°C, a stirring time of 25 min, and a stirring speed of 400 r / min, stirring and mixing to obtain an emulsion mixture; S4, placing the emulsion mixture prepared in S3 into a rotary evaporator with a power of 2.5 kW, a rotary evaporation temperature of 30°C, a rotary evaporation time of 1.5 h, a rotary evaporation speed of 120 r / min, and placing it into a freeze dryer with a power of 0.8 kW, a freeze drying temperature of 4°C, a freeze drying time of 12 h, and freeze drying to obtain amplification-promoting nanoparticles; S5. Place the amplification-promoting nanoparticles prepared by S4 into the culture medium matrix prepared by S1, and place it in an ultrasonic agitator with a power of 1.8 kW, an ultrasonic frequency of 15 kHz, a stirring speed of 20,000 r / min, a stirring temperature of 25°C, and a stirring time of 10 min to obtain a rapid amplification culture medium.

[0029] In S1, the mass fraction of the human AB serum in the culture medium matrix is ​​10%, the volume ratio of the human AB serum to the X-VIVO15 serum-free culture medium is 1:9, the mass fraction of the cytokine combination in the culture medium matrix is ​​8%, including: 300 U / mL IL-2, 300 U / mL IL-15, 300 U / mL IL-21, 200 pg / mL epidermal growth factor, 30 ng / mLCD3 antibody, 30 ng / mL CD28 antibody and 20 ng / mL LAG-3 antibody, the mass fraction of insulin in the culture medium matrix is ​​2.5%, and the weight ratio of the lentinan, sodium pyruvate and insulin is 2:1:1.

[0030] In S2, the mass fraction of eicosapentaenoic acid in anhydrous ethanol is 34%.

[0031] In S3, the mass fraction of the polysorbate in ultrapure water is 28%.

[0032] Example 2: This example provides a method for preparing tumor infiltrating lymphocytes, which specifically includes the following steps: Step 1: Place fresh lung cancer tumor tissue in a 10 cm culture medium, wash twice with PBS buffer, remove the necrotic area and connective tissue on the fresh tumor tissue with a sterile scalpel, and cut into 2 mm 3 of tumor mass; Step 2: Put a keratinase solution with a mass concentration of 1.3%, a 0.6% myristoyl coenzyme A solution, and a 1.4% N-myristoyl transferase solution into a reactor with a power of 2.3 kW, the reaction temperature is 30°C, the reaction time is 3.4 h, the reaction speed is 200 r / min, and after the reaction, put it into a freeze-spray dryer with a power of 2.6 kW, the freeze-spray drying temperature is -30°C, the freeze-spray drying particle size is 250 mm, and the freeze-spray drying time is 1.3 h to obtain a modified keratinase; Step 3: Place the modified keratinase prepared in step 2, fibronectin, elastase, and RPMI 1640 culture medium in a magnetic stirrer with a power of 1.3 kW, stir at a speed of 320 r / min, a temperature of 23° C., and a stirring time of 13 min, and stir and mix to obtain a preliminary enzymatic solution; Step 4: Put the preliminary enzymatic hydrolysate prepared in step 3 and the tumor block prepared in step 1 into a centrifuge tube, and place it in a water bath with a power of 2.2 kW, a water bath temperature of 37° C., a water bath time of 1.1 h, and perform enzymatic hydrolysis in a water bath to obtain loose tissue; Step 5: Rinse the loose tissue prepared in step 4 with 0.9% sterile saline for 3 times, add proteinase-K and RPMI 1640 culture medium, put into a centrifuge tube, and place in a water bath with a power of 2.2 kW. The water bath temperature is 20°C and the water bath time is 1.5 h. After low-temperature enzymolysis, filter with a 200-mesh stainless steel filter to obtain a cell suspension; Step 6: Place the cell suspension prepared in step 5 into a centrifuge with a power of 2.7 kW, centrifuge at a speed of 12000 r / min, a centrifuge temperature of 2°C, and a centrifuge time of 13 min, discard the supernatant, and obtain the precipitated cells; Step 7: After resuspending the precipitated cells prepared in step 6 with RPMI-1640 medium, mix with lymphocyte separation solution using a 25 mL pipette, put into a centrifuge with a power of 2.8 kW, the centrifugal speed is 12000 r / min, the centrifugal temperature is 2°C, the centrifugal time is 13 min, and after centrifugation, use a 10 mL pipette to absorb the upper cell layer to obtain a suspension of tumor-infiltrating lymphocytes after separation; Step 8: Inoculate the isolated tumor-infiltrating lymphocyte suspension prepared in step 7 into a rapid expansion medium, place it in a CO2 constant temperature and humidity incubator, and change the medium once a week under the conditions of a constant temperature culture temperature of 37° C. and a CO2 volume fraction of 5%. Culture for 16 days to obtain a tumor-infiltrating lymphocyte culture medium; Step 9: Place the tumor infiltrating lymphocyte culture medium prepared in step 8 into a centrifuge with a power of 2.8 kW, a centrifugal speed of 12000 r / min, a centrifugal temperature of 2°C, and a centrifugal time of 13 min. After centrifugation, discard the supernatant to obtain tumor infiltrating lymphocytes.

[0033] In step 1, the pH of the PBS buffer is 7.4.

[0034] In step 2, the volume ratio of the keratinase solution, myristoyl-CoA solution and N-myristoyl transferase solution is 1.5:1.1:1.

[0035] In step three, the weight ratio of the modified keratinase, fibronectin and elastase is 5:2.5:1, and the mass fraction of the elastase in the RPMI-1640 culture medium is 0.8%.

[0036] In step 5, the mass fraction of the proteinase-K in the RPMI-1640 medium is 1.9%.

[0037] In step eight, the method for preparing the rapid expansion medium specifically comprises the following steps: S1. Put X-VIVO 15 serum-free medium, human AB serum, cytokine composition, insulin, Lentinan, and sodium pyruvate into a stirrer with a power of 1.4 kW, stir at a temperature of 24° C., stir for 22 min, and stir at a speed of 400 r / min to mix and obtain a culture medium matrix; S2, placing eicosapentaenoic acid and anhydrous ethanol in a 1.4 kW stirrer at a stirring temperature of 26°C, a stirring time of 16 min, and a stirring speed of 300 r / min, and fully dissolving to obtain an eicosapentaenoic acid solution; S3, placing polysorbate, ultrapure water, and the culture medium matrix prepared in S1 into the eicosapentaenoic acid solution prepared in S2, stirring at a temperature of 40°C, a stirring time of 28 min, and a stirring speed of 500 r / min, stirring and mixing to obtain an emulsion mixture; S4, placing the emulsion mixture prepared in S3 into a rotary evaporator with a power of 2.5 kW, the rotary evaporation temperature is 33°C, the rotary evaporation time is 1.8 h, the rotary evaporation speed is 130 r / min, and then placing it into a freeze dryer with a power of 1.1 kW, the freeze drying temperature is 4°C, the freeze drying time is 14 h, and freeze drying is performed to obtain amplification-promoting nanoparticles; S5. Place the amplification-promoting nanoparticles prepared by S4 into the culture medium matrix prepared by S1, and place it in an ultrasonic agitator with a power of 1.8 kW, an ultrasonic frequency of 15 kHz, a stirring speed of 20,000 r / min, a stirring temperature of 25°C, and a stirring time of 10 min to obtain a rapid amplification culture medium.

[0038] In S1, the mass fraction of the human AB serum in the culture medium matrix is ​​10%, the volume ratio of the human AB serum to the X-VIVO15 serum-free culture medium is 1:9, the mass fraction of the cytokine combination in the culture medium matrix is ​​9%, including: 300 U / mL IL-2, 300 U / mL IL-15, 300 U / mL IL-21, 200 pg / mL epidermal growth factor, 30 ng / mLCD3 antibody, 30 ng / mL CD28 antibody and 20 ng / mL LAG-3 antibody, the mass fraction of insulin in the culture medium matrix is ​​3.2%, and the weight ratio of the lentinan, sodium pyruvate and insulin is 2.5:2:1.

[0039] In S2, the mass fraction of eicosapentaenoic acid in anhydrous ethanol is 35%.

[0040] In S3, the mass fraction of the polysorbate in ultrapure water is 32%.

[0041] Example 3: This example provides a method for preparing tumor infiltrating lymphocytes, which specifically comprises the following steps: Step 1: Place fresh gastric cancer tumor tissue in a 10 cm culture medium, wash it three times with PBS buffer, remove the necrotic area and connective tissue on the fresh tumor tissue with a sterile scalpel, and cut it into 3 mm 3 of tumor mass; Step 2: Put a keratinase solution with a mass concentration of 1.4%, a 0.7% myristoyl-CoA solution, and a 1.5% N-myristoyl transferase solution into a reactor with a power of 2.4 kW, the reaction temperature is 37°C, the reaction time is 3.6 h, the reaction speed is 200 r / min, and after the reaction, put it into a freeze-spray dryer with a power of 2.8 kW, the freeze-spray drying temperature is -30°C, the freeze-spray drying particle size is 250 mm, and the freeze-spray drying time is 1.4 h to obtain a modified keratinase; Step 3: Place the modified keratinase prepared in step 2, fibronectin, elastase, and RPMI 1640 culture medium in a magnetic stirrer with a power of 1.4 kW, stir at a speed of 350 r / min, a temperature of 25°C, and a stirring time of 15 min, and stir and mix to obtain a preliminary enzymatic solution; Step 4: Put the preliminary enzymatic hydrolysate prepared in step 3 and the tumor block prepared in step 1 into a centrifuge tube, and place it in a water bath with a power of 2.2 kW, a water bath temperature of 37° C., a water bath time of 1.3 h, and perform enzymatic hydrolysis in a water bath to obtain loose tissue; Step 5: Rinse the loose tissue prepared in step 4 with 0.9% sterile saline for 3 times, add proteinase-K and RPMI 1640 culture medium, put into a centrifuge tube, and place in a water bath with a power of 2.2 kW. The water bath temperature is 20°C and the water bath time is 2 h. After low-temperature enzymolysis, filter with a 200-mesh stainless steel filter to obtain a cell suspension; Step 6: Place the cell suspension prepared in step 5 into a centrifuge with a power of 2.9 kW, centrifuge at a speed of 12000 r / min, a centrifuge temperature of 2°C, and a centrifuge time of 15 min, discard the supernatant, and obtain the precipitated cells; Step 7: After resuspending the precipitated cells prepared in step 6 with RPMI-1640 medium, mix with lymphocyte separation solution using a 25 mL pipette, put into a centrifuge with a power of 2.9 kW, the centrifugal speed is 12000 r / min, the centrifugal temperature is 2°C, the centrifugal time is 15 min, and after centrifugation, use a 10 mL pipette to absorb the upper cell layer to obtain a suspension of tumor-infiltrating lymphocytes after separation; Step 8: Inoculate the isolated tumor-infiltrating lymphocyte suspension prepared in step 7 into a rapid expansion medium, place it in a CO2 constant temperature and humidity incubator, and change the medium twice a week under the conditions of a constant temperature and humidity culture temperature of 37°C and a CO2 volume fraction of 5%. Culture for 18 days to obtain a tumor-infiltrating lymphocyte culture medium; Step 9: Place the tumor infiltrating lymphocyte culture medium prepared in step 8 into a centrifuge with a power of 2.9 kW, a centrifugal speed of 12000 r / min, a centrifugal temperature of 2°C, and a centrifugal time of 15 min. After centrifugation, discard the supernatant to obtain tumor infiltrating lymphocytes.

[0042] In step 1, the pH of the PBS buffer is 7.4.

[0043] In step 2, the volume ratio of the keratinase solution, myristoyl-CoA solution and N-myristoyl transferase solution is 3:1.2:1.

[0044] In step three, the weight ratio of the modified keratinase, fibronectin and elastase is 6:3:1, and the mass fraction of the elastase in the RPMI-1640 culture medium is 0.9%.

[0045] In step 5, the mass fraction of the proteinase-K in the RPMI-1640 medium is 2.1%.

[0046] In step eight, the method for preparing the rapid expansion medium specifically comprises the following steps: S1. Put X-VIVO 15 serum-free culture medium, human AB serum, cytokine composition, insulin, Lentinan, and sodium pyruvate into a stirrer with a power of 1.6 kW, stir at a temperature of 28° C., stir for 26 min, and stir at a speed of 500 r / min to mix and obtain a culture medium matrix; S2, placing eicosapentaenoic acid and anhydrous ethanol in a 1.6 kW stirrer at a stirring temperature of 28°C, a stirring time of 18 min, and a stirring speed of 400 r / min, until the eicosapentaenoic acid solution is fully dissolved; S3, placing polysorbate, ultrapure water, and the culture medium matrix prepared in S1 into the eicosapentaenoic acid solution prepared in S2, stirring at a temperature of 40°C, a stirring time of 30 min, and a stirring speed of 600 r / min, stirring and mixing to obtain an emulsion mixture; S4, placing the emulsion mixture prepared in S3 into a rotary evaporator with a power of 2.5 kW, a rotary evaporation temperature of 35°C, a rotary evaporation time of 2 h, a rotary evaporation speed of 150 r / min, placing it into a freeze dryer with a power of 1.2 kW, a freeze drying temperature of 4°C, a freeze drying time of 16 h, and freeze drying to obtain amplification-promoting nanoparticles; S5. Place the amplification-promoting nanoparticles prepared by S4 into the culture medium matrix prepared by S1, and place it in an ultrasonic agitator with a power of 1.8 kW, an ultrasonic frequency of 15 kHz, a stirring speed of 20,000 r / min, a stirring temperature of 25°C, and a stirring time of 10 min to obtain a rapid amplification culture medium.

[0047] In S1, the mass fraction of the human AB serum in the culture medium matrix is ​​10%, the volume ratio of the human AB serum to the X-VIVO15 serum-free culture medium is 1:9, the mass fraction of the cytokine combination in the culture medium matrix is ​​10%, including: 300 U / mL IL-2, 300 U / mL IL-15, 300 U / mL IL-21, 200 pg / mL epidermal growth factor, 30 ng / mLCD3 antibody, 30 ng / mL CD28 antibody and 20 ng / mL LAG-3 antibody, the mass fraction of the insulin in the culture medium matrix is ​​5%, the mass fraction of the insulin in the culture medium matrix is ​​3.8%, and the weight ratio of the lentinan, sodium pyruvate and insulin is 3:3:1.

[0048] In S2, the mass fraction of eicosapentaenoic acid in anhydrous ethanol is 37%.

[0049] In S3, the mass fraction of the polysorbate in ultrapure water is 36%.

[0050] Comparative Example 1: This comparative example provides a method for separating tumor infiltrating lymphocytes, comprising the following steps: Step 1: Place fresh liver cancer tumor tissue in a 10 cm culture medium, wash twice with PBS buffer, remove the necrotic area and connective tissue on the fresh tumor tissue with a sterile scalpel, and cut into 1 mm 3 of tumor mass; Step 2: Place fibronectin, elastase, and RPMI 1640 culture medium in a 1.2 kW magnetic stirrer at a stirring speed of 300 r / min, a stirring temperature of 20°C, and a stirring time of 10 min, and stir and mix to obtain a preliminary enzymatic solution; Step 3: Put the preliminary enzymatic hydrolysate prepared in step 2 and the tumor block prepared in step 1 into a centrifuge tube, and place it in a water bath with a power of 2.2 kW, a water bath temperature of 37° C., a water bath time of 0.8 h, and perform enzymatic hydrolysis in the water bath to obtain loose tissue; Step 4: Rinse the loose tissue prepared in step 3 twice with 0.9% sterile saline, put it into a centrifuge tube with collagenase II and RPMI 1640 culture medium, and place it in a water bath with a power of 2.2 kW. The water bath temperature is 40°C and the water bath time is 1 h. After low-temperature enzymolysis, filter it with a stainless steel filter with a pore size of 200 to obtain a cell suspension; Step 5: Place the cell suspension prepared in step 4 into a centrifuge with a power of 2.6 kW, centrifuge at a speed of 12000 r / min, a centrifuge temperature of 2°C, and a centrifuge time of 10 min, discard the supernatant, and obtain the precipitated cells; Step 6: After resuspending the precipitated cells prepared in step 6 with RPMI-1640 medium, mix with lymphocyte separation solution using a 25 mL pipette, put into a centrifuge with a power of 2.6 kW, the centrifugal speed is 12000 r / min, the centrifugal temperature is 2°C, the centrifugal time is 10 min, and after centrifugation, use a 10 mL pipette to absorb the upper cell layer to obtain a suspension of tumor-infiltrating lymphocytes after separation; Step 7: Inoculate the isolated tumor-infiltrating lymphocyte suspension prepared in step 6 into a rapid expansion medium, place it in a CO2 constant temperature and humidity incubator, and culture it at a constant temperature of 37°C and a CO2 volume fraction of 5%. Change the medium once a week and culture it for 14 days to obtain a tumor-infiltrating lymphocyte culture medium. Step 8: Place the tumor-infiltrating lymphocyte culture medium prepared in step 7 into a centrifuge with a power of 2.6 kW, a centrifugal speed of 12000 r / min, a centrifugal temperature of 2°C, and a centrifugal time of 10 min. After centrifugation, discard the supernatant to obtain tumor-infiltrating lymphocytes.

[0051] In step 1, the pH of the PBS buffer is 7.4.

[0052] In step 2, the weight ratio of the fibronectin to the elastase is 2:1, and the mass fraction of the elastase in the RPMI-1640 culture medium is 0.4%.

[0053] In step 4, the mass fraction of collagenase II in the RPMI-1640 culture medium is 1.7%.

[0054] The components, component contents, and method steps of the rapid expansion medium used are the same as those in Example 1.

[0055] Comparative Example 2: This comparative example provides a method for separating tumor infiltrating lymphocytes, comprising the following steps: Among them, the components, component contents, and method steps of the separation method steps 1 to 9 are the same as those of Example 1; The method for preparing the rapid expansion medium specifically comprises the following steps: S1. Place X-VIVO 15 serum-free medium, human AB serum, cytokine composition, insulin, Lentinan, and sodium pyruvate into a 1.2 kW agitator at a stirring temperature of 20°C, a stirring time of 18 min, and a stirring speed of 300 r / min to mix and obtain a rapid expansion medium.

[0056] In S1, the mass fraction of the human AB serum in the culture medium matrix is ​​10%, the volume ratio of the human AB serum to the X-VIVO15 serum-free culture medium is 1:9, the mass fraction of the cytokine combination in the culture medium matrix is ​​8%, including: 300 U / mL IL-2, 300 U / mL IL-15, 300 U / mL IL-21, 200 pg / mL epidermal growth factor, 30 ng / mLCD3 antibody, 30 ng / mL CD28 antibody and 20 ng / mL LAG-3 antibody, the mass fraction of insulin in the culture medium matrix is ​​2.5%, and the weight ratio of the lentinan, sodium pyruvate and insulin is 2:1:1.

[0057] Experimental Example 1: Proliferation rate and purity determination test.

[0058] The experimental steps for determining the proliferation rate and purity of tumor infiltrating lymphocytes prepared in Examples 1-3 of the present invention are as follows: (1) The tumor infiltrating lymphocytes obtained in Example 1-3 and Comparative Example 1-2 were washed twice with RPMI-1640, centrifuged at 800 r / min for 3 min, and resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and IL-2 at a final concentration of 1000 IU / mL to adjust the cell concentration to 1×10 6 / mL for activation culture, placed in a 37°C, 5% CO2 incubator, observed the cells under an inverted microscope, and performed subculture according to the growth conditions; (2) MTT colorimetric method to detect the proliferation rate of tumor infiltrating lymphocytes obtained in Example 1-3 and Comparative Example 1-2: 80 μL of cells with a concentration of 1×10 6Each group of samples with a concentration of 5 mg / mL was inoculated in a 96-well plate, with 3 replicate wells in each group. On the 0th and 4th days of activation culture, 20 mL of 5 mg / mL diphenyl tetrazolium bromide was added to each well and cultured for 4 h. 100 mL of the triple dissolution solution was added to each well and placed in a 37°C, 5% CO2 incubator overnight. The optical density OD value of each well at 490 nm was measured with an enzyme-labeled instrument, and the cell proliferation rate was calculated according to the formula: cell proliferation rate % = (OD t -OD0) / OD0×100%,OD t is the OD value of the tumor-infiltrating lymphocyte group on the 4th day of activated culture, and OD0 is the OD value of the tumor-infiltrating lymphocyte group on the 0th day of activated culture; (3) Flow cytometry was used to detect the purity of tumor infiltrating lymphocytes obtained in Example 1-3 and Comparative Example 1-2: The samples of each group after activation and culture for 14 days were prepared into a single cell suspension with a density of 1×10 5 / mL, add fluorescently labeled CD3, CD4 and CD8 monoclonal antibodies, and place in the dark for 20 min; add 3 mL of cell washing solution to the mixed suspension, centrifuge at 1000 r / min for 2 min, remove the unbound monoclonal antibodies in the supernatant, and repeat the washing twice, wash the precipitated cells once with PBS solution with a pH value of 7.4, centrifuge at 1000 r / min for 5 min, fix the precipitated cells with 1% paraformaldehyde, and then detect the cell purity by flow cytometry; (4) The tumor infiltrating lymphocytes obtained in Example 1-3 and Comparative Example 1-2 were mixed with lymphocyte cryopreservation solution and stored at -150°C for 14 days, then taken out, thawed at 37°C, centrifuged at 800 r / min for 3 min, and resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum and IL-2 at a final concentration of 1000 IU / mL to adjust the cell concentration to 1×10 6 / mL for activation culture, placed in a 37°C, 5% CO2 incubator, observed the cells under an inverted microscope, and performed subculture according to the growth conditions; (5) MTT colorimetric assay for the proliferation rate of tumor infiltrating lymphocytes obtained in Example 1-3 and Comparative Example 1-2 after storage in step (4): 80 μL of cells with a cell concentration of 1×10 6 / mL of each group of samples were inoculated in a 96-well plate, with 3 replicate wells in each group. On the 0th and 4th days of activation culture, 20 mL of 5 mg / mL diphenyltetrazolium bromide was added to each well and cultured for 4 h. Then 100 mL of triple dissolution solution was added to each well and the plates were placed in a 37°C, 5% CO2 incubator overnight. The optical density OD value of each well at 490 nm was measured with an enzyme-labeled instrument to calculate the cell proliferation rate after storage.

[0059] Result analysis: Figure 1 This is a microscope observation image of the activated tumor infiltrating lymphocytes prepared in Example 1 of the present invention on the 4th day of culture. Figure 2 The result graph of the proliferation rate of tumor infiltrating lymphocytes described in Experimental Example 1 of the present invention is shown in the figure. As shown in the figure, the proliferation rates of the tumor infiltrating lymphocytes obtained in Examples 1-3 at the 4th day of activation culture were 59.2%, 62.1%, and 63.2% respectively by MTT colorimetry. After the mixed lymphocyte cryopreservation solution was stored at -150°C for 14 days, the proliferation rates of the tumor infiltrating lymphocytes obtained in Examples 1-3 after storage were 59.1%, 61.8%, and 62.6% respectively by MTT colorimetry. The proliferation rate of the tumor infiltrating lymphocytes obtained in Comparative Example 1 at the 4th day of activation culture was 41.3% by MTT colorimetry. After the mixed lymphocyte cryopreservation solution was stored at -150°C for 14 days, the proliferation rate of the tumor infiltrating lymphocytes obtained in Comparative Example 1 at the 4th day of activation culture was 40.1% by MTT colorimetry. The tumor infiltrating lymphocytes obtained in Comparative Example 2 were activated and cultured for 4 days. The proliferation rate at d was 46.1%. After the mixed lymphocyte cryopreservation solution was stored at -150°C for 14 days, the proliferation rate of the tumor infiltrating lymphocytes obtained in Comparative Example 2 after storage by MTT colorimetry was 44.4% at 4 days after activation culture. Figure 3 This is a graph showing the purity of tumor infiltrating lymphocytes described in Experimental Example 1 of the present invention. As shown in the figure, the purity of the tumor infiltrating lymphocytes obtained in Examples 1-3 after 14 days of activation and culture was 99.2%, 99.6%, and 99.7%, respectively, detected by flow cytometry. The purity of the tumor infiltrating lymphocytes obtained in Comparative Example 1 after 14 days of activation and culture was 89.5%. The purity of the tumor infiltrating lymphocytes obtained in Comparative Example 2 after 14 days of activation and culture was 98.6%, indicating that the method for preparing tumor infiltrating lymphocytes of the present invention can produce tumor infiltrating lymphocytes with high activity, rapid proliferation and growth, and high cell activity after cryopreservation.

[0060] Experimental Example 2: Breast cancer tumor inhibition rate determination test.

[0061] The experimental steps for determining the inhibition rate of breast cancer tumors by tumor infiltrating lymphocytes prepared in Examples 1-3 of the present invention are as follows: MTT colorimetric method was used to detect the tumor killing ability of tumor infiltrating lymphocytes obtained in Example 1-3 and Comparative Example 1-2: The concentration of DOX breast cancer 4T1-Luc cells was adjusted to 1×10 5 / mL, 80 μL was inoculated in a 96-well plate and placed in a 37°C, 5% CO2 incubator for 24 h; 50 μL of each group of tumor infiltrating lymphocytes activated for 14 days was added according to the ratio of tumor infiltrating lymphocytes to DOX breast cancer 4T1-Luc cells of 50:1, and a single tumor infiltrating lymphocyte group and a single DOX breast cancer 4T1-Luc cell group were set up at the same time, with 3 replicate wells in each group; after culturing in a 37°C, 5% CO2 incubator for 24 h, 20 μL of 5 mg / mL MTT was added to each well and continued to be cultured for 4 h. 100 μL of triple dissolution solution was added to each well, and placed in a 37°C, 5% CO2 incubator overnight. The optical density OD value of each well at 490 nm was measured with an enzyme marker, and the breast cancer tumor cell killing rate was calculated according to the following formula: Killing rate % = [(OD T +OD E )-OD E+T ] / OD T × 100%, where OD T is the OD value of the DOX breast cancer 4T1-Luc cell group alone, OD E is the OD value of the tumor infiltrating lymphocyte group alone, OD E+T is the OD value of the experimental group.

[0062] Result analysis: Figure 4 This is a result diagram of the tumor infiltrating lymphocytes described in Experimental Example 2 of the present invention for the inhibition rate of breast cancer tumors. As shown in the figure, the tumor infiltrating lymphocytes obtained in Examples 1-3 were detected by MTT colorimetry, and were placed in a 37°C, 5% CO2 incubator for 24 h, and MTT was added to each well for continued culture for 4 h. After adding the triple dissolution solution and placing it overnight, the inhibition rates for DOX breast cancer 4T1-Luc cells were 62.1%, 64.2%, and 66.8%, respectively. The tumor infiltrating lymphocytes obtained in Comparative Example 1 were detected by MTT colorimetry, and were placed in a 37°C, 5% CO2 incubator for 24 h, and MTT was added to each well for continued culture for 4 h. After adding the triple dissolution solution and placing it overnight, the inhibition rate for DOX breast cancer 4T1-Luc cells was 47.3%. The tumor infiltrating lymphocytes obtained in Comparative Example 2 were detected by MTT colorimetry, and were placed in a 37°C, 5% CO2 incubator for 24 h, and MTT was added to each well for continued culture for 4 h. After adding the triple dissolution solution and leaving it overnight, the inhibition rate for DOX breast cancer 4T1-Luc cells was 53.4%, indicating that the method for preparing tumor infiltrating lymphocytes of the present invention has high activity of tumor infiltrating lymphocytes and good anti-breast tumor activity.

[0063] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0064] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. The present invention provides a method for preparing tumor infiltrating lymphocytes, characterized in that: The specific steps include: Step 1: Wash the fresh tumor tissue with PBS buffer for 2-3 times and cut it into tumor pieces with a sterile scalpel; Step 2, mixing keratinase with myristoyl-CoA and N-myristoyl transferase, and then freeze-spray drying to obtain modified keratinase; Step 3, mixing the modified keratinase prepared in step 2 with fibronectin, elastase, and RPMI-1640 culture medium to obtain a preliminary enzymatic solution; Step 4: Put the preliminary enzymatic hydrolysate prepared in step 3 and the tumor block prepared in step 1 into a centrifuge tube, and perform enzymatic hydrolysis in a water bath to obtain loose tissue; Step 5: Rinse the loose tissue prepared in step 4 with 0.9% sterile saline for 2-3 times, add proteinase-K and RPMI-1640 culture medium, put into a centrifuge tube, perform enzymatic hydrolysis, and filter with a 200-mesh stainless steel filter to obtain a cell suspension; Step 6: Centrifuge the cell suspension prepared in step 5 and discard the supernatant to obtain precipitated cells; Step 7: Resuspend the precipitated cells prepared in step 6 with RPMI-1640 medium, mix with lymphocyte separation solution using a 25 mL pipette, centrifuge and use a 10 mL pipette to aspirate the upper cell layer to obtain a separated tumor-infiltrating lymphocyte suspension; Step 8, inoculating the separated tumor infiltrating lymphocyte suspension prepared in step 7 into a rapid expansion medium for culturing to obtain a tumor infiltrating lymphocyte culture fluid; Step 9: Centrifuge the tumor infiltrating lymphocyte culture medium prepared in step 8, discard the supernatant, and obtain tumor infiltrating lymphocytes.

2. The method for preparing tumor infiltrating lymphocytes according to claim 1, characterized in that: In step eight, the method for preparing the rapid expansion medium specifically comprises the following steps: S1, mixing X-VIVO 15 serum-free medium, human AB serum, cytokine combination, insulin, Lentinan, and sodium pyruvate to obtain a culture medium matrix; S2, fully dissolving eicosapentaenoic acid in anhydrous ethanol to obtain an eicosapentaenoic acid solution; S3, adding polysorbate, ultrapure water, and the culture medium matrix prepared in S1 into the eicosapentaenoic acid solution prepared in S2, stirring and mixing to obtain an emulsion mixture; S4, rotary evaporating the emulsion mixture prepared in S3, and then freeze-drying it to obtain amplification-promoting nanoparticles; S5. Place the amplification-promoting nanoparticles prepared in S4 into the culture medium matrix prepared in S1, and mix them to obtain a rapid amplification culture medium.

3. The present invention also proposes the use of tumor infiltrating lymphocytes prepared by the method for preparing tumor infiltrating lymphocytes according to any one of claims 1 to 2 in a drug for treating breast cancer, characterized in that: The specific steps include: I. Thawing the stored tumor-infiltrating lymphocytes at 37° C., centrifuging them, and removing the precipitate to obtain the tumor-infiltrating lymphocytes; II. Mixing the tumor infiltrating lymphocytes obtained in step I with pharmaceutically acceptable excipients to obtain a drug for treating breast cancer.

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