TIL cell culture medium and TIL cell culture method

Abstract

The invention discloses a TIL cell culture medium and a TIL cell culture method, an activation and proliferation culture medium of TIL cells comprises the following components: a T cell serum-free culture medium, IL-2, platelet lysate and gentamicin, a CD3 monoclonal antibody, a CD28 monoclonal antibody and phytolectin PHA-P are added for cell activation, and then the activation and proliferation culture medium is used for activation and proliferation culture. Wherein CD3 and CD28 antibodies are used in combination to stimulate TIL cells and simulate the double-signal effect of in-vivo TIL cell activation, meanwhile, PHA-P is utilized, by means of the characteristics of PHA-P, small gonorrhea cells are stimulated in vitro to be converted into gonorrhea cells with immunocompetence, division of the gonorrhea cells is promoted, and the proliferation capacity of the TIL cells is improved through the synergistic effect of PHA-P and IL-2; platelet lysate is used for replacing plasma of a patient and is used as an efficient culture supplement for supporting TIL cell growth; finally, more TIL cells are obtained through the technology, and the number of immune cells required by clinical effective treatment can be sufficiently met.

Description

Technical Field

[0001] The patent of this invention belongs to the field of biotechnology, and specifically relates to TIL cell culture medium and TIL cell culture method. Background Art

[0002] TIL (Tumor Infiltrating Lymphocytes) are heterogeneous lymphocytes in the tumor stroma, including T cells and NK cells. Some of these lymphocytes are T cells that target tumor-specific mutant antigens. They are the immune cells with the strongest ability to penetrate deep into the enemy and are considered to be a specific immune response of the body to tumor cells. In recent years, TILs therapy has been proven to be able to be used to treat melanoma, cervical cancer, lung cancer, sarcoma and ovarian cancer. It also shows great potential in malignant tumors such as colorectal cancer and breast cancer, fully demonstrating the potential of TIL as a cellular immunotherapy in tumor treatment; therefore, isolating TIL from tumor tissue and activating and amplifying it in vitro can be used as a practical and effective specific immunotherapy for tumor patients.

[0003] TIL is mainly obtained through four methods: tissue block culture, enzyme digestion, mechanical dissociation, and fine needle aspiration. The existing TIL cell culture method mainly uses IL-2 as the main stimulating factor, and human AB serum as a nutritional supplement for co-culture. However, under such culture conditions, the cells cannot maintain a low level of differentiation, and their proliferation ability and long-term persistence in the body are low, which cannot meet the number of immune cells required for effective clinical treatment.

[0004] Therefore, this article proposes a TIL cell culture medium and a TIL cell culture method suitable for large-scale expansion of TIL cells with cytotoxic activity. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a TIL cell culture medium and a TIL cell culture method suitable for amplifying a large number of TIL cells with cytotoxic activity.

[0006] In order to achieve the above technical effects, the present invention provides a TIL cell culture medium, including an activation culture medium and a proliferation culture medium; The components of the activation medium include T cell serum-free medium, 8%-12% platelet lysate, 900-1100U / ml interleukin-2, and 30-50U / ml gentamicin; The components of the proliferation culture medium include T cell serum-free culture medium, 4%-6% platelet lysate, 4800-5200U / ml interleukin-2, and 30-50U / ml gentamicin.

[0007] Preferably according to the present invention, the volume percentage of platelet lysate in the activation culture medium is 5%, 1000U / ml interleukin-2, and 40U / ml gentamicin.

[0008] Preferably according to the present invention, the volume percentage of platelet lysate in the proliferation culture medium is 10%, 5000U / ml interleukin-2, and 40U / ml gentamicin.

[0009] The present invention also provides a TIL cell culture method, comprising using the TIL cell culture medium of the present invention, and the method specifically comprises the following steps: S1. Immune cell separation: After enzymatic digestion of tumor tissue, immune cells are obtained using lymphocyte separation fluid, or immune cells in malignant pleural effusion and ascites are directly obtained using lymphocyte separation fluid; Furthermore, the method for isolating immune cells from tumor tissue in S1 specifically comprises the following steps: S111. The tumor tissue was washed with physiological saline to remove fat tissue, necrotic tissue and bleeding clots, and minced with tissue scissors. Five volumes of trypsin / EDTA digestion solution were added, followed by 90-110 U / ml type IV collagenase, 4-6 U / ml DNase I and 75-85 U / ml gentamicin. The mixture was mixed with the tumor tissue and then placed in a shaker at 37°C at 100 rpm / min for 3 h for digestion. S112, adding an equal volume of physiological saline to dilute the tissue digestion solution in a ratio of 1:1, filtering the solution with a cell sieve, centrifuging the filtrate, discarding the supernatant, and washing the precipitate with physiological saline by centrifugation; S113, taking a centrifuge tube and adding lymphocyte separation solution, resuspending the cell precipitate with physiological saline and slowly adding it to the upper layer of the lymphocyte separation solution, and centrifuging slowly up and down; S114. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the diluent layer, the second layer is the ring-shaped milky white target cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the tissue miscellaneous cell layer. Carefully pipette the second layer into the centrifuge tube, add physiological saline and centrifuge, discard the supernatant, and the precipitate is the immune cells.

[0010] Furthermore, the method for isolating immune cells from malignant pleural effusions and ascites in S1 specifically comprises the following steps: S121, malignant pleural effusion and ascites were added to centrifuge tubes and centrifuged, and the supernatant was discarded; S122, take a centrifuge tube and add lymphocyte separation solution, resuspend the cell precipitate with physiological saline and slowly add it to the upper layer of lymphocyte separation solution, and centrifuge slowly up and down; S123. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the diluent layer, the second layer is the ring-shaped milky white target cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the tissue miscellaneous cell layer. Carefully pipette the second layer into the centrifuge tube, add physiological saline and centrifuge, discard the supernatant, and the precipitate is the immune cells.

[0011] S2. Adhesion to remove residual tumor cells: first prepare activation culture medium, then culture the immune cells in the activation culture medium for 3 days to allow the tumor cells to adhere to the wall, take the supernatant and centrifuge to obtain the immune cells, and discard the rest to remove residual tumor cells; Furthermore, the method for removing residual tumor cells by adhesion in S2 specifically comprises the following steps: S201, activation medium configuration: T cell serum-free medium with 8%-12% platelet lysate, 900-1100 U / ml interleukin-2, and 30-50 U / ml gentamicin added; S202. Resuspend the cells in activation medium at a density of 2×106 cells / ml and inoculate them into a 6-well plate. Culture them at 37°C and 5% CO2 for 3 days. Gently shake the culture plate, transfer the non-adherent cell suspension to a centrifuge tube, and centrifuge. Discard the supernatant.

[0012] S3. TIL cell activation: After counting the cell pellet, resuspend it in activation medium at a density of 2×106 cells / ml, add 90-110ug / ml of plant lectin PHA-P, 900-1100ng / ml of CD3 monoclonal antibody, and 900-1100ng / ml of CD28 monoclonal antibody to induce TIL cell activation and proliferation; culture at 37°C and 5% CO2 for 3 days; Furthermore, the substance components added to the activation medium of S3 are: 100ug / ml of phytohemagglutinin PHA-P, 1000ng / ml of CD3 monoclonal antibody, and 1000ng / ml of CD28 monoclonal antibody.

[0013] S4. After TIL cells are activated, TIL cell activation culture is performed: activation medium is supplemented on the third day of culture, and then once every three days. After rehydration, the TIL cell culture density is not less than 1×106 cells / ml; S5. When the cells begin to grow exponentially, proliferation culture of TIL cells is performed using a proliferation medium, wherein the method specifically comprises the following steps: S501, proliferation medium configuration: T cell serum-free medium with 4%-6% platelet lysate, 4800-5200U / ml interleukin-2, and 30-50U / ml gentamicin; S502, TIL cells were switched to proliferation medium for rehydration after they began to grow exponentially, and cultured for 40-50 days. When the cells no longer grew exponentially, the cells were harvested; S6. Finally, cell detection is performed, and the method specifically comprises the following steps: S601, take the cell culture supernatant to test for sterility, mycoplasma, and endotoxin; S602. Take cells and use flow cytometry to detect T lymphocyte typing CD3, CD4, CD8 and their proportions.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly: The present invention uses CD3 and CD28 antibodies to stimulate TIL cells, simulates the dual signal effect of TIL cell activation in vivo, and simultaneously utilizes PHA-P, which has the characteristics of promoting peripheral blood lymphocyte mitosis and inducing interferon secretion, to stimulate small lymphocytes to transform into lymphocytes with immune activity in vitro and promote their division, and enhances the proliferation ability of TIL cells through the synergistic effect of PHA-P and IL-2; and also uses platelet lysate to replace patient plasma, as a high-efficiency culture supplement that can effectively support the growth of TIL cells; finally, the present invention obtains more TIL cells with killing activity through the steps of activation, activation culture, and proliferation culture, which is sufficient to meet the number of immune cells required for effective clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for describing the embodiment are briefly introduced below. Those skilled in the art can also obtain other drawings based on these drawings without paying any creative work.

[0016] Figure 1 This is a morphological diagram of unactivated TIL cells in an embodiment of the present invention; Figure 2 This is the morphology of TIL cells after activation in the embodiment of the present invention; Figure 3 This is a diagram of TIL cell subpopulation analysis in an embodiment of the present invention; Figure 4 This is a diagram of TIL cell killing analysis in an embodiment of the present invention; Figure 5 This is a graph showing the expansion multiples of TIL cells in an embodiment of the present invention; DETAILED DESCRIPTION

[0017] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0018] The prior art in the present invention has the following problems: because the existing TIL cell culture method mainly uses IL-2 as the main stimulating factor, and is supplemented with human AB serum as a nutritional supplement for co-culture, but under such culture conditions, the cells cannot maintain a low level of differentiation, and their proliferation ability and long-term persistence in the body are low, and cannot meet the number of immune cells required for effective clinical treatment.

[0019] Therefore, the inventors provide a TIL cell culture medium, including an activation culture medium and a proliferation culture medium; The components of the activation medium include T cell serum-free medium, 8%-12% platelet lysate, 900-1100U / ml interleukin-2, and 30-50U / ml gentamicin; The components of the proliferation culture medium include T cell serum-free culture medium, 4%-6% platelet lysate, 4800-5200U / ml interleukin-2, and 30-50U / ml gentamicin.

[0020] Further preferably, the volume percentage of platelet lysate in the activation medium is 5%, 1000 U / ml interleukin-2, and 40 U / ml gentamicin.

[0021] Further preferably, the volume percentage of platelet lysate in the proliferation medium is 10%, 5000U / ml interleukin-2, and 40U / ml gentamicin.

[0022] The present invention also provides a TIL cell culture method, comprising using the TIL cell culture medium of the present invention, and the method specifically comprises the following steps: S1. Immune cell separation: After enzymatic digestion of tumor tissue, immune cells are obtained using lymphocyte separation fluid, or immune cells in malignant pleural effusion and ascites are directly obtained using lymphocyte separation fluid; Furthermore, the method for isolating immune cells from tumor tissue in S1 specifically comprises the following steps: S111. The tumor tissue was washed three times with normal saline to remove fat tissue, necrotic tissue and blood clots, and minced into 1 mm³ with tissue scissors. Five volumes of trypsin / EDTA digestion solution were added, followed by type IV collagenase (90-110 U / ml), DNase I (4-6 U / ml), and gentamicin (75-85 U / ml). The mixture was mixed with the tumor tissue and then placed in a shaker at 37°C at 100 rpm / min for 3 h for digestion. S112, dilute the tissue digestion solution with an equal volume of physiological saline in a ratio of 1:1, filter the solution with a 70um cell sieve, centrifuge the filtrate at 500g for 5 minutes, discard the supernatant, and wash the precipitate twice with physiological saline by centrifugation; S113, take a 15 ml centrifuge tube and add 2 ml of lymphocyte separation solution, resuspend the cell precipitate with 2 ml of normal saline and slowly add it to the upper layer of lymphocyte separation solution, and centrifuge at 500g for 20 min. S114. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the diluent layer, the second layer is the ring-shaped milky white target cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the tissue miscellaneous cell layer. Carefully pipette the second layer into a 15ml centrifuge tube, add 5ml of physiological saline, centrifuge at 500g for 5 minutes, discard the supernatant, and the precipitate is the immune cell.

[0023] Furthermore, the method for isolating immune cells from malignant pleural effusions and ascites in S1 specifically comprises the following steps: S121, malignant pleural and ascites were added to a 50 ml centrifuge tube and centrifuged at 500 g for 5 min, and the supernatant was discarded; S122. Take a 15 ml centrifuge tube and add 2 ml of lymphocyte separation solution. Resuspend the cell pellet with 2 ml of normal saline and slowly add it to the upper layer of lymphocyte separation solution. Centrifuge at 500 g for 20 min. S123. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the diluent layer, the second layer is the ring-shaped milky white target cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the tissue miscellaneous cell layer. Carefully pipette the second layer into a 15ml centrifuge tube, add 5ml of physiological saline, centrifuge at 500g for 5 minutes, discard the supernatant, and the precipitate is the immune cells.

[0024] S2. Adhesion to remove residual tumor cells: first prepare activation culture medium, then culture the immune cells in the activation culture medium for 3 days to allow the tumor cells to adhere to the wall, take the supernatant and centrifuge to obtain the immune cells, and discard the rest to remove residual tumor cells; Furthermore, the method for removing residual tumor cells by adhesion in S2 specifically comprises the following steps: S201, activation medium configuration: T cell serum-free medium with 8%-12% platelet lysate, 900-1100 U / ml interleukin-2, and 30-50 U / ml gentamicin added; S202. Resuspend the cells in activation medium at a density of 2×106 cells / ml and inoculate them into a 6-well plate. Culture them at 37°C and 5% CO2 for 3 days. Gently shake the culture plate, pipette the non-adherent cell suspension into a 15-ml centrifuge tube, centrifuge at 500 g for 5 min, and discard the supernatant.

[0025] S3, TIL cell activation: After counting the cell pellet, resuspend it in activation medium at a density of 2×106 cells / ml, add plant lectin PHA-P (90-110ug / ml), CD3 monoclonal antibody (900-1100ng / ml), CD28 monoclonal antibody (900-1100ng / ml) to induce TIL cell activation and proliferation; culture at 37°C, 5% CO2 for 3 days; Furthermore, the substance components added to the activation medium of S3 are: plant lectin PHA-P (100ug / ml), CD3 monoclonal antibody (1000ng / ml), and CD28 monoclonal antibody (1000ng / ml).

[0026] like Figure 1 to Figure 2 As shown, T cells do not aggregate when not activated, but aggregate after being activated by CD3, CD28 monoclonal antibodies and cytokine IL-2; in the early stage of culture, cells have no obvious growth, which is the growth inhibition period, and in the later stage, they begin to proliferate rapidly, increasing from 2 million cells at the beginning to 3.2 billion.

[0027] S4. After TIL cells are activated, TIL cell activation culture is performed: activation medium is supplemented on the third day of culture, and then once every three days. After rehydration, the TIL cell culture density is not less than 1×106 cells / ml; S5. When the cells begin to grow exponentially, proliferation culture of TIL cells is performed using a proliferation medium, wherein the method specifically comprises the following steps: S501, proliferation medium configuration: T cell serum-free medium with 4%-6% platelet lysate, 4800-5200U / ml interleukin-2, and 30-50U / ml gentamicin; S502, TIL cells were switched to proliferation medium for rehydration after they began to grow exponentially, and cultured for 40-50 days. When the cells no longer grew exponentially, the cells were harvested; S6. Finally, cell detection is performed, and the method specifically comprises the following steps: S601, take the cell culture supernatant to test for sterility, mycoplasma, and endotoxin; S602. Take cells and use flow cytometry to detect T lymphocyte typing and proportion (CD3, CD4, CD8).

[0028] like Figure 3 As shown, after 50 days of activation culture, cells were taken for flow cytometry detection of lymphocyte subsets. The results showed that T lymphocytes accounted for 82.5%, of which helper T lymphocytes (CD3+CD4+) accounted for 25.5%, and cytotoxic T lymphocytes (CD3+CD8+) accounted for 75.5%, which was consistent with the characteristics of TIL cell clustering.

[0029] like Figure 4 As shown, successfully cultured TIL cells and tumor cells were co-cultured at an effector-target ratio of 4:1 for 24 hours, and the tumor cell killing was detected by flow cytometry. The results showed that 6.7% of tumor cells died and 42.2% of them underwent early apoptosis, indicating that TIL cells have good killing effect on tumor cells.

[0030] In the process of studying TIL cell culture, in order to test the effects of platelet lysate and PHA-P on TIL cell culture, the inventors also conducted a comparative experiment to verify: Example

[0031] The components of T cell serum-free medium were added at each stage according to the following scheme:

[0032] After isolating immune cells from tumor tissue or malignant pleural effusion, PBMCs were resuspended in the above activation medium at a density of 2×106 / ml and placed in a 37°C, 5% CO2 cell culture incubator for 3 days to remove tumor cells. On the 3rd day, suspended immune cells were separated and added with cell activation medium at a density of 2×106 / ml, and placed in a 37°C, 5% CO2 cell culture incubator for culture until the 15th day. The activation medium was replaced every 3 days to maintain a cell density of 1-3×106 / mL. After the 18th day, the proliferation culture was replaced and continued until TIL cells were obtained after the 45th day of culture.

[0033] Comparative Example 1 The T cell serum-free culture medium was added with various components according to the following scheme at each stage, wherein PHA-P was removed at the cell activation stage, and the rest was the same as in Example 1 (as shown in the following table):

[0034] After isolating immune cells from tumor tissue or malignant pleural effusion, PBMCs were resuspended in the above activation medium at a density of 2×106 / ml and placed in a 37°C, 5% CO2 cell culture incubator for 3 days to remove tumor cells. On the 3rd day, suspended immune cells were separated and added with cell activation medium at a density of 2×106 / ml, and placed in a 37°C, 5% CO2 cell culture incubator for culture until the 15th day. The activation medium was replaced every 3 days to maintain a cell density of 1-3×106 / mL. After the 18th day, the proliferation culture was replaced and continued until TIL cells were obtained after the 45th day of culture.

[0035] Comparative Example 2 The T cell serum-free medium was added with various components according to the following scheme at each stage, wherein the activation medium reduced the platelet lysate to 5%, and the proliferation medium removed the platelet lysate, and the rest was the same as in Example 1 (as shown in the following table):

[0036] After isolating immune cells from tumor tissue or malignant pleural effusion, PBMCs were resuspended in the above activation medium at a density of 2×106 / ml and placed in a 37°C, 5% CO2 cell culture incubator for 3 days to remove tumor cells. On the 3rd day, suspended immune cells were separated and added with cell activation medium at a density of 2×106 / ml, and placed in a 37°C, 5% CO2 cell culture incubator for culture until the 15th day. The activation medium was replaced every 3 days to maintain a cell density of 1-3×106 / mL. After the 18th day, the proliferation culture was replaced and continued until TIL cells were obtained after the 45th day of culture.

[0037] Results comparison: Grouping Starting cell number Harvested cell number Amplification factor Example 4.74×106 1.36×109 287 Comparative Example 1 4.74×106 1.19×109 251 Comparative Example 2 4.74×106 1.02×109 215 As shown in the table above Figure 5 As shown, if TIL cells are cultured according to the culture method and culture medium ratio in the technical solution disclosed in the present invention, TIL cells with a greater expansion multiple can be obtained.

[0038] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0039] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. TIL cell culture medium, including activation culture medium and proliferation culture medium, characterized in that: The components of the activation medium include T cell serum-free medium, 8%-12% platelet lysate, 900-1100U / ml interleukin-2, and 30-50U / ml gentamicin; The components of the proliferation culture medium include T cell serum-free culture medium, 4%-6% platelet lysate, 4800-5200U / ml interleukin-2, and 30-50U / ml gentamicin.

2. The TIL cell culture medium according to claim 1, characterized in that: The volume percentage of platelet lysate in the activation culture medium is 5%, 1000U / ml interleukin-2, and 40U / ml gentamicin.

3. The TIL cell culture medium according to claim 1, characterized in that: The volume percentage of platelet lysate in the proliferation culture medium is 10%, 5000U / ml interleukin-2, and 40U / ml gentamicin.

4. The TIL cell culture method according to any one of claims 1 to 3, comprising using the TIL cell culture medium, characterized in that: The method specifically comprises the following steps: S1. Immune cell separation: After enzymatic digestion of tumor tissue, immune cells are obtained using lymphocyte separation fluid, or immune cells in malignant pleural effusion and ascites are directly obtained using lymphocyte separation fluid; S2. Adhesion to remove residual tumor cells: first prepare activation culture medium, then culture the immune cells in the activation culture medium for 3 days to allow the tumor cells to adhere to the wall, take the supernatant and centrifuge to obtain the immune cells, and discard the rest to remove residual tumor cells; S3. TIL cell activation: After counting the cell pellet, resuspend it in activation medium at a density of 2×106 cells / ml, add 90-110ug / ml of plant lectin PHA-P, 900-1100ng / ml of CD3 monoclonal antibody, and 900-1100ng / ml of CD28 monoclonal antibody to induce TIL cell activation and proliferation; culture at 37°C and 5% CO2 for 3 days; S4. After TIL cells are activated, TIL cell activation culture is performed: activation medium is supplemented on the third day of culture, and then once every three days. After the replenishment, the TIL cell culture density is not less than 1×106 cells / ml; S5. When the cells begin to grow exponentially, proliferation culture of TIL cells is performed using a proliferation medium, wherein the method specifically comprises the following steps: S501, proliferation medium configuration: T cell serum-free medium with 4%-6% platelet lysate, 4800-5200U / ml interleukin-2, and 30-50U / ml gentamicin; S502, TIL cells were switched to proliferation medium for rehydration after they began to grow exponentially, and cultured for 40-50 days. When the cells no longer grew exponentially, the cells were harvested; S6. Finally, cell detection is performed, and the method specifically comprises the following steps: S601, take the cell culture supernatant to test for sterility, mycoplasma, and endotoxin; S602. Take cells and use flow cytometry to detect T lymphocyte typing CD3, CD4, CD8 and their proportions.

5. The TIL cell culture method according to claim 4, characterized in that: The method for isolating immune cells from tumor tissue in S1 specifically comprises the following steps: S111. The tumor tissue was washed with physiological saline to remove fat tissue, necrotic tissue and bleeding clots, and minced with tissue scissors. Five volumes of trypsin / EDTA digestion solution were added, followed by 90-110 U / ml type IV collagenase, 4-6 U / ml DNase I and 75-85 U / ml gentamicin. The mixture was mixed with the tumor tissue and then placed in a shaker at 37°C at 100 rpm / min for 3 h for digestion. S112, adding an equal volume of physiological saline to dilute the tissue digestion solution in a ratio of 1:1, filtering the solution with a cell sieve, centrifuging the filtrate, discarding the supernatant, and washing the precipitate with physiological saline by centrifugation; S113, taking a centrifuge tube and adding lymphocyte separation solution, resuspending the cell precipitate with physiological saline and slowly adding it to the upper layer of the lymphocyte separation solution, and centrifuging slowly up and down; S114. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the diluent layer, the second layer is the ring-shaped milky white target cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the tissue miscellaneous cell layer. Carefully pipette the second layer into the centrifuge tube, add physiological saline and centrifuge, discard the supernatant, and the precipitate is the immune cells.

6. The TIL cell culture method according to claim 4, characterized in that: The method for isolating immune cells from malignant pleural effusion in S1 specifically comprises the following steps: S121, malignant pleural effusion and ascites were added to centrifuge tubes and centrifuged, and the supernatant was discarded; S122, take a centrifuge tube and add lymphocyte separation solution, resuspend the cell precipitate with physiological saline and slowly add it to the upper layer of lymphocyte separation solution, and centrifuge slowly up and down; S123. After centrifugation, the centrifuge tube is divided into four layers from top to bottom: the first layer is the diluent layer, the second layer is the ring-shaped milky white target cell layer, the third layer is the transparent separation liquid layer, and the fourth layer is the tissue miscellaneous cell layer. Carefully pipette the second layer into the centrifuge tube, add physiological saline and centrifuge, discard the supernatant, and the precipitate is the immune cells.

7. The TIL cell culture method according to claim 4, characterized in that: The method for removing residual tumor cells by adherence in S2 specifically comprises the following steps: S201, activation medium configuration: T cell serum-free medium with 8%-12% platelet lysate, 900-1100 U / ml interleukin-2, and 30-50 U / ml gentamicin added; S202. Resuspend the cells in activation medium at a density of 2×106 cells / ml and inoculate them into a 6-well plate. Culture them at 37°C and 5% CO2 for 3 days. Gently shake the culture plate, transfer the non-adherent cell suspension to a centrifuge tube, and centrifuge. Discard the supernatant.

8. The TIL cell culture method according to claim 4, characterized in that: The material components added to the activation culture medium of S3 are: 100ug / ml of phytohemagglutinin PHA-P, 1000ng / ml of CD3 monoclonal antibody, and 1000ng / ml of CD28 monoclonal antibody.

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