An isolation culture method for malignant ascites derived TIL cells

A technology for separating and culturing cells, which is applied in the field of separating and culturing TIL cells, can solve the problems of complicated operations, reduced cell viability, and shortened time spent on separating and culturing TIL cells, and achieves strong tumor killing activity, simple separation and culturing methods, and increased targeting. sexual effect

Inactive Publication Date: 2016-12-21
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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  • An isolation culture method for malignant ascites derived TIL cells
  • An isolation culture method for malignant ascites derived TIL cells
  • An isolation culture method for malignant ascites derived TIL cells

Examples

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Embodiment 1

[0037] The malignant ascites 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 ascites twice with D-Hanks solution, add appropriate amount of PBS buffer solution (containing 0.5% BSA, 2 mmol EDTA, pH 7.2) to precipitate cells, separate mononuclear cells with Ficoll lymphocyte separation medium, count and 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 place in a refrigerator at 2-8 degrees Ce...

Embodiment 2

[0058] The malignant ascites 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 ascites 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 isolation culture method for malignant ascites derived TIL cells. At least one of PD-1, LAG-3 and TIM-3 is adopted as a label for isolating tumor-specific T lymphocytes from the TIL cells. The tumor-specific T lymphocytes are separated from the TIL cell mass by combining an immunomagnetic bead technique, and then subjected to in-vitro efficient amplification to obtain the tumor-specific TIL cells having high tumor killing activity. The isolation culture of the tumor-specific TIL cells is simpler in operation procedures, obviously shortened in isolation culture time, low in cost and convenient to popularize and apply. The tumor killing activity of the obtained tumor-specific TIL cells is 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 ascites. Background technique [0002] Tumor-infiltrating lymphocytes (TIL) are lymphocytes present in tumor tissues, tumor-draining lymph nodes, and cancerous ascites. 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 Treatment of Patien...

Claims

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Application Information

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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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