Isolated culture method of malignant pleural effusion-sourced TIL cells

A technology for separation and cultivation of malignant pleural effusion, applied in the field of separation and cultivation of TIL cells, which can solve the problems of complicated operations, shortened time spent on the separation and cultivation of TIL cells, and decreased cell viability, etc., and achieves strong tumor killing activity, increased pertinence, and separation and cultivation The effect of simple method

Inactive Publication Date: 2016-11-16
英普乐孚生物技术(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The object of the present invention is to provide a method for separating and cultivating TIL cells, which will eliminate tumors with tumor Tumor-specific T lymphocytes with specific killing activity are separated from the TIL cell population and subjected to massive expansion and culture. powerful

Method used

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  • Isolated culture method of malignant pleural effusion-sourced TIL cells
  • Isolated culture method of malignant pleural effusion-sourced TIL cells
  • Isolated culture method of malignant pleural effusion-sourced TIL cells

Examples

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Embodiment 1

[0037] The malignant pleural effusion of a liver cancer patient (female, 48 years old) is now taken as the material for preparing tumor-specific TIL cells. The isolation and culture process of tumor-specific TIL cells is as follows:

[0038] Step 1: Preparation of single cell suspension.

[0039] 1.2 Wash the pleural effusion twice with D-Hanks solution, add an appropriate amount of PBS buffer solution (containing 0.5% BSA, 2 mmol EDTA, pH 7.2) to the precipitated cells, and separate the mononuclear cells with Ficoll lymphocyte separation medium. After counting, resuspend in PBS buffer solution, The final single-cell suspension of tumor tissue was obtained.

[0040] Step 2: Separate and obtain tumor-specific TIL cells by immunomagnetic bead technology.

[0041] 2.1 Adjust the final cell concentration obtained in step 1 to 2.5×10 per 1.0ml of buffer solution 8 cells.

[0042] 2.2 Add 100 ul of Fc receptor blocker (Miltenyi Biotec, product number 130-059-901), mix well, and p...

Embodiment 2

[0058] The malignant pleural effusion of a colorectal cancer patient (male, 53 years old) is now taken as the material for preparing tumor-specific TIL cells. The isolation and culture process of tumor-specific TIL cells is as follows:

[0059] Step 1 and step 3 are with embodiment 1;

[0060] The operation process of step 2 is the same as that of Example 1, the antibody added in step 2.4 is different, it is Anti-TIM-3-APC (Miltenyi Biotec, catalog number 130-098-936); the magnetic beads added in step 2.6 are different, are anti-APC magnetic beads (Miltenyi Biotec, Cat. No. 130-090-855).

Embodiment 3

[0062] The malignant pleural effusion of a colorectal cancer patient (female, 58 years old) was used as the material for preparing tumor-specific TIL cells. The isolation and culture process of tumor-specific TIL cells was as follows:

[0063] Step 1 and step 3 are with embodiment 1;

[0064] The operation process of step 2 is the same as that of Example 1, the differences are: the antibody added in step 2.4 is different, it is a mouse anti-human LAG-3 antibody (R&D SYSTEMS, article number: MAB23193, IgG1); the magnetic field added in step 2.6 Beads are different, anti-mouse IgG1 magnetic beads (Miltenyi Biotec, Cat. No. 130-047-101)

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Abstract

The invention relates to an isolated culture method of malignant pleural effusion-sourced TIL cells. The isolated culture method comprises the following steps: using at least one of PD-1, LAG-3 and TIM-3 as a marker for isolating tumor-specific T lymphocytes from TIL cells, isolating the tumor-specific T lymphocytes from a TIL cell population in combination with an immunomagnetic bead technology, and performing in-vitro efficient amplification to obtain tumor-specific TIL cells with high tumor-killing activities. By the isolated culture method, the isolated culture of the tumor-specific TIL cells is simpler in operational process and the isolated culture time is significantly shortened; the isolated culture method is low in cost and convenient to popularize and apply; in addition, the tumor-killing activities of the isolated tumor-specific TIL cells are stronger.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and relates to a method for separating and culturing TIL cells (tumor infiltrating lymphocytes) derived from malignant pleural effusion. Background technique [0002] Tumor-infiltrating lymphocytes (TIL) are lymphocytes present in tumor tissues, tumor-draining lymph nodes, and cancerous pleural effusions. Compared with peripheral blood lymphocytes, TIL cells contain a higher proportion of activated tumor-specific T lymphocytes , after this type of lymphocytes are isolated, expanded, and activated in vitro, their killing activity against tumor cells is enhanced, and they can be used for adoptive cell immunotherapy of tumors. [0003] Good clinical results have been achieved using this technique. Rosenberg et al. used whole body radiotherapy + chemotherapy + TIL cells to treat malignant melanoma, and the total effective rate reached 72% (Rosenberg SA1, Dudley ME.Adoptive Cell Therapy for the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0636A61K35/17C12N2501/2302C12N2501/50C12N2501/51C12N2501/515C12N2509/00
Inventor 武宁谢志明
Owner 英普乐孚生物技术(上海)有限公司
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