Isolated culturing method for tumor-specific TIL cells

A tumor-specific, separation and culture technology, applied in the field of separation and culture of tumor-specific TIL cells, can solve the problems of complicated operation, reduced cell viability, and shortened time spent in the separation and culture of TIL cells, and achieves strong tumor killing activity, separation and culture. Simple method, increase targeted effect

Inactive Publication Date: 2015-09-30
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-

Method used

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  • Isolated culturing method for tumor-specific TIL cells
  • Isolated culturing method for tumor-specific TIL cells
  • Isolated culturing method for tumor-specific TIL cells

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Embodiment 1

[0039] The surgically resected tumor tissue of a patient with liver cancer (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:

[0040] Step 1: Preparation of tumor tissue single cell suspension.

[0041] 1.1 Take tumor tissue pieces, remove normal tissue and necrotic tissue, cut into small pieces of tissue with a diameter of 2mm with scissors, and immerse these small tissue pieces in collagenase containing 2.0mg / ml (SIGMA, product number: C9722), 0.5mg / ml Hyaluronidase (SIGMA, product number: H3506), (10-0.01) 0.1 mg / ml DNase (SIGMA, product number: D5319) in serum-free RPMI 1640 medium, and incubated overnight with slow shaking. Filter with a 100um nylon filter to remove cell aggregates and obtain a single cell suspension.

[0042] 1.2 Wash the single cell suspension twice with D-Hanks solution, add appropriate amount of PBS buffer solution (containing 0.5% B...

Embodiment 2

[0061] The surgically resected tumor tissue of a colorectal cancer patient (male, 53 years old) is now used as the material for preparing tumor-specific TIL cells. The isolation and culture process of tumor-specific TIL cells is as follows:

[0062] Step 1 and step 3 are with embodiment 1;

[0063] 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

[0065] The surgically resected tumor tissue 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:

[0066] Step 1 and step 3 are with embodiment 1;

[0067] 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 culturing method for tumor-specific TIL cells. According to the method, one or more from PD-1, LAC-3 and TIM 3 are utilized for serving as markers for separating tumor specific T-lymphocytes in TIL cells, the immunomagnetic bead technology is combined, the tumor specific T-lymphocytes are separated from a TIL cell mass, then in-vitro efficient amplification is conducted, and the tumor-specific TIL cells with high tumor killing activity are obtained. By means of the isolated culturing method for the tumor-specific TIL cells, the separated culturing operation procedure of the tumor-specific TIL cells is made to be simpler, the separated culturing time is obviously shortened, the cost is low, the application and popularization are facilitated, and moreover through the separation, the obtained tumor-specific TIL cells are higher in tumor cell killing activity.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and relates to a method for separating and culturing tumor-specific TIL cells (tumor infiltrating lymphocytes). 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 Patients with Metastat...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
Inventor 谢志明武宁崔文浩郑蓉蓉
Owner 英普乐孚生物技术(上海)有限公司
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