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Method for utilizing malignant hydrothorax and ascitic fluid containing clots to prepare TIL cells having high killing activity

A technology of killing activity and clotting, applied in the field of cell culture, can solve the problems of affecting the separation of mononuclear cells, difficult to screen tumor-specific TIL cells, affecting the purity of tumor-specific TIL cells and anti-tumor effect, etc.

Active Publication Date: 2016-12-14
QINGDAO CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent document CN104946589A uses PD-1, LAG-3 or TIM-3 antibody-coated magnetic beads to screen the mononuclear cells isolated from malignant pleural / ascites fluid, and then stimulates the screening cell expansion with OKT3 and IL-2. The method cannot solve the problem of insufficient activation and scarcity of tumor-specific TIL cells in malignant pleural / ascites caused by the low expression of MHC-Ⅰ / Ⅱ molecules in tumor cells and the existence of an immunosuppressive microenvironment, and it is difficult to screen out high-abundance TIL cells. Moreover, since Tregs and other tumor antigen non-specific TIL cells also express molecules such as PD-1, LAG-3 or TIM-3, they will also enter the screening cells along with the immunomagnetic beads And get a lot of amplification, which will affect the purity and anti-tumor effect of tumor-specific TIL cells in the cultured final product
During density gradient centrifugation, these clots will interfere with stratification and affect the separation of mononuclear cells; at the same time, because the clots sink to the bottom of the centrifuge tube with high specific gravity cell components such as red blood cells and granulocytes, they are discarded. This may result in a massive loss of TIL cells
[0008] However, there is no report in the prior art on the use of clot-containing malignant pleural effusion to prepare a large number of TIL cells with high killing activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0073] (1) Collection of clot-containing malignant pleural effusion: under sterile conditions, collect 1000 mL of clot-containing malignant pleural effusion caused by lung cancer, and add 15,000 U of heparin;

[0074] (2) Separation of free cells and clots in malignant pleural effusion containing clots: use a nylon microporous filter with a pore size of 70 μm to filter the malignant pleural effusions containing clots to separate the clots and free cells in malignant pleural effusions Separated to obtain free cell-A;

[0075] (3) Decomposition of the clot and release of free cells: the clot was placed in AIM-V complete medium-I containing 750 U / mL IFN-γ, 55 U / mL IL-2 and 3% inactivated serum in 37°C, 5% CO 2 Cultivate for 3 days under the condition of saturated humidity and filter to obta...

Embodiment 2

[0083] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using clot-containing malignant pleural effusion includes the following steps:

[0084] (1) Collection of clot-containing malignant ascites: under sterile conditions, collect 1200 mL of clot-containing malignant ascites caused by gastric cancer, and add 18,000 U of heparin;

[0085] (2) Separation of free cells and clots in malignant ascites containing clots: use a nylon microporous filter with a pore size of 70 μm to filter the malignant ascites containing clots to separate the clots and free cells in malignant ascites. Separated to obtain free cell-A;

[0086] (3) Decomposition of clot and release of free cells: place the clot in AIM-V complete medium-I containing 500 U / mL IFN-γ, 100 U / mL IL-2 and 3% inactivated serum At 37°C, 5% CO 2 Cultivate for 4 days under the condition of saturated humidity and filter to obtain free cell-B;

[0087] (4) Separation of mononuc...

Embodiment 3

[0094] In this embodiment, the method for preparing a large amount of TIL cells with high killing activity by using clot-containing malignant pleural effusion includes the following steps:

[0095] (1) Collection of clot-containing malignant pleural effusion: under sterile conditions, collect 1000 mL of clot-containing malignant pleural effusion caused by lung cancer, and add 15,000 U of heparin;

[0096] (2) Separation of free cells and clots in malignant pleural effusion containing clots: use a nylon microporous filter with a pore size of 70 μm to filter the malignant pleural effusions containing clots to separate the clots and free cells in malignant pleural effusions Separated to obtain free cell-A;

[0097] (3) Decomposition of clot and release of free cells: place the clot in AIM-V complete medium-I containing 500 U / mL IFN-γ, 100 U / mL IL-2 and 3% inactivated serum At 37°C, 5% CO 2 Cultivate for 5 days under the condition of saturated humidity and filter to obtain free ...

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Abstract

The invention relates to a method for utilizing a malignant hydrothorax and ascitic fluid containing clots to prepare TIL cells having high killing activity. The method comprises the steps that the TIL cells (190.1 + / - 46.7) * 108 (n = 12) can be obtained when culture is performed for 21-28 days, and cell proliferation is up to 182.5 + / - 46.8 times, wherein CD3+ cells account for about 98.55% + / - 2.24%, CD3+CD8+ cells account for about 76.11% + / - 6.88%, CD3+CD4+ cells account for about 24.09% + / - 6.73%, CD3+CD6+ cells account for about 53.36% + / - 9.47%, CD4+CD25+FoxP3+Tregs cells account for only 2.87% + / - 1.65%, the tumor cell killing activity can be up to 60.57% + / - 5.34% (4h51Cr release method, the effect / target = 40:1). Compared with the situation when clots are not treated, the obtained TIL cells number can be increased by about 30%.

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 clot-containing malignant pleural effusion. 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), toget...

Claims

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Application Information

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