A method for preparing a large amount of til cells with high killing activity by using malignant pleural effusion

Abstract

The invention relates to a method for preparing high-killing-activity TIL cells in batches from malignant pleuroperitoneal fluid. The method comprises the steps that (338.6+ / -134.2)*10<8>(n=25) TIL cells can be obtained when culture is performed for 21-28 d, the cells are amplified by 179.6+ / -24.2 times, wherein, CD3+cells account for 98.61%+ / -3.22%, CD3+CD8+cells account for 74.56%+ / -5.19%, CD3+CD4+cells account for 27.42%+ / -6.35%, CD3+CD56+cells account for 51.88%+ / -7.49%, and CD4+CD25+FoxP3+ Tregs cells only account for 3.27%+ / -1.75%. The killing activity for tumor cells can reach 66.54%+ / -5.18% (according to a 4h51Cr release method, the effect / target ratio is equal to 40:1).

Description

technical field

[0001] The invention belongs to the field of cell culture, and in particular relates to a method for preparing a large amount of TIL cells with high killing activity by using malignant pleural ascites. Background technique

[0002] Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of lymphocytes present in tumor tissue or tumor regional lymph nodes, including CD3 preactivated by tumor antigens. + CD4 + Th cells (T helper lymphocytes) and CD3 + CD8 + CTL cells (cytotoxic T lymphocytes) can specifically recognize and kill tumor cells in an MHC-restricted manner; a certain amount of tumor antigen non-specific CD3 + CD4 + or CD3 + CD8 + T cells; small amount of CD3 - CD56 + NK cells (natural killers) kill tumor cells in a non-MHC-restricted manner; in addition, they also contain a certain amount of CD4 with immunosuppressive effects + CD25 + FoxP3 + Tregs (regulatory T cells) and iDCs (immature dendritic cells), together with highly e...

Examples

Embodiment 1

[0063] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using malignant pleural effusion comprises the following steps:

[0064] (1) Collection of malignant pleural effusion: under sterile conditions, collect 1000 mL of malignant pleural effusion caused by lung cancer, and add 15,000 U of heparin;

[0065] (2) Separation of mononuclear cells: transfer the malignant pleural effusion into a centrifuge tube, centrifuge at 500×g for 5 minutes, and discard the supernatant; Centrifuge at 1600×g for 15 minutes on the Ficoll layering solution of 1.077;

[0066] (3) Washing of mononuclear cells: collect mononuclear cells (including lymphocytes, DC cells and tumor cells) on the interface of Ficoll layering solution, add AIM-V serum-free medium, pipette, mix, and centrifuge at 300×g 5min, discard the supernatant; repeat the following steps twice: add AIM-V serum-free medium to resuspend the cells, pipette, mix, centrifuge at 300×g fo...

Embodiment 2

[0072] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using malignant pleural effusion comprises the following steps:

[0073] (1) Collection of malignant ascites: under sterile conditions, collect 1200 mL of malignant ascites caused by gastric cancer, and add 18,000 U of heparin;

[0074] (2) Separation of mononuclear cells: transfer the malignant ascites into a centrifuge tube, centrifuge at 500×g for 5 min, and discard the supernatant; Centrifuge at 1600×g for 15 minutes on the Ficoll layering solution of 1.076;

[0075] (3) Washing of mononuclear cells: collect mononuclear cells (including lymphocytes, DC cells and tumor cells) on the interface of Ficoll layering solution, add AIM-V serum-free medium, pipette, mix, and centrifuge at 300×g 5min, discard the supernatant; repeat the following steps twice: add AIM-V serum-free medium to resuspend the cells, pipette, mix, centrifuge at 300×g for 5min, discard the supernat...

Embodiment 3

[0081] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using malignant pleural effusion comprises the following steps:

[0082] (1) Collection of malignant pleural effusion: under sterile conditions, collect 1600 mL of malignant pleural effusion caused by lung cancer, and add 20,000 U of heparin;

[0083] (2) Separation of mononuclear cells: transfer the malignant pleural effusion into a centrifuge tube, centrifuge at 500×g for 5 minutes, and discard the supernatant; Centrifuge at 1600×g for 15 minutes on the Ficoll layering solution of 1.078;

[0084] (3) Washing of mononuclear cells: collect mononuclear cells (including lymphocytes, DC cells and tumor cells) on the interface of Ficoll layering solution, add AIM-V serum-free medium, pipette, mix, and centrifuge at 300×g 5min, discard the supernatant; repeat the following steps twice: add AIM-V serum-free medium to resuspend the cells, pipette, mix, centrifuge at 300×g fo...

technical field

[0001] The invention belongs to the field of cell culture, and in particular relates to a method for preparing a large amount of TIL cells with high killing activity by using malignant pleural ascites. Background technique

[0002] Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of lymphocytes present in tumor tissue or tumor regional lymph nodes, including CD3 preactivated by tumor antigens. + CD4 + Th cells (T helper lymphocytes) and CD3 + CD8 + CTL cells (cytotoxic T lymphocytes) can specifically recognize and kill tumor cells in an MHC-restricted manner; a certain amount of tumor antigen non-specific CD3 + CD4 + or CD3 + CD8 + T cells; small amount of CD3 - CD56 + NK cells (natural killers) kill tumor cells in a non-MHC-restricted manner; in addition, they also contain a certain amount of CD4 with immunosuppressive effects + CD25 + FoxP3 + Tregs (regulatory T cells) and iDCs (immature dendritic cells), together with highly e...

Examples

Embodiment 1

[0063] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using malignant pleural effusion comprises the following steps:

[0064] (1) Collection of malignant pleural effusion: under sterile conditions, collect 1000 mL of malignant pleural effusion caused by lung cancer, and add 15,000 U of heparin;

[0065] (2) Separation of mononuclear cells: transfer the malignant pleural effusion into a centrifuge tube, centrifuge at 500×g for 5 minutes, and discard the supernatant; Centrifuge at 1600×g for 15 minutes on the Ficoll layering solution of 1.077;

[0066] (3) Washing of mononuclear cells: collect mononuclear cells (including lymphocytes, DC cells and tumor cells) on the interface of Ficoll layering solution, add AIM-V serum-free medium, pipette, mix, and centrifuge at 300×g 5min, discard the supernatant; repeat the following steps twice: add AIM-V serum-free medium to resuspend the cells, pipette, mix, centrifuge at 300×g fo...

Embodiment 2

[0072] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using malignant pleural effusion comprises the following steps:

[0073] (1) Collection of malignant ascites: under sterile conditions, collect 1200 mL of malignant ascites caused by gastric cancer, and add 18,000 U of heparin;

[0074] (2) Separation of mononuclear cells: transfer the malignant ascites into a centrifuge tube, centrifuge at 500×g for 5 min, and discard the supernatant; Centrifuge at 1600×g for 15 minutes on the Ficoll layering solution of 1.076;

[0075] (3) Washing of mononuclear cells: collect mononuclear cells (including lymphocytes, DC cells and tumor cells) on the interface of Ficoll layering solution, add AIM-V serum-free medium, pipette, mix, and centrifuge at 300×g 5min, discard the supernatant; repeat the following steps twice: add AIM-V serum-free medium to resuspend the cells, pipette, mix, centrifuge at 300×g for 5min, discard the supernat...

Embodiment 3

[0081] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using malignant pleural effusion comprises the following steps:

[0082] (1) Collection of malignant pleural effusion: under sterile conditions, collect 1600 mL of malignant pleural effusion caused by lung cancer, and add 20,000 U of heparin;

[0083] (2) Separation of mononuclear cells: transfer the malignant pleural effusion into a centrifuge tube, centrifuge at 500×g for 5 minutes, and discard the supernatant; Centrifuge at 1600×g for 15 minutes on the Ficoll layering solution of 1.078;

[0084] (3) Washing of mononuclear cells: collect mononuclear cells (including lymphocytes, DC cells and tumor cells) on the interface of Ficoll layering solution, add AIM-V serum-free medium, pipette, mix, and centrifuge at 300×g 5min, discard the supernatant; repeat the following steps twice: add AIM-V serum-free medium to resuspend the cells, pipette, mix, centrifuge at 300×g fo...