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

A kind of killing activity, chest and abdomen technology, applied in the field of cell culture, can solve the problem of low specific killing efficiency of tumor cells, and achieve the effect of high killing activity

Active Publication Date: 2019-10-22
QINGDAO CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For this reason, what the present invention aims to solve is the technical problem in the prior art that the TIL cells isolated and cultured from malignant pleural effusion have low specific killing efficiency on tumor cells, and further provide a method for preparing a large amount of TIL cells with high killing activity by using malignant pleural effusion. Methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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...

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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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
Inventor 解西河魏晓芳郭庆明
Owner QINGDAO CENT HOSPITAL
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