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Method for amplifying tumor killer T cells in tumor infiltrating lymphocytes

A lymphocyte and tumor infiltration technology, applied in the field of biomedicine, can solve the problems of slow growth, different expansion efficiency of tumor-killing T cells, inability to activate and maintain the killing power of T cells, etc., to improve consistency and increase expansion efficiency. and tumor-killing activity

Pending Publication Date: 2020-04-21
北京科途医学科技有限公司
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Problems solved by technology

[0003] At present, the culture of tumor-infiltrating lymphocytes is mainly carried out in a conventional two-dimensional culture environment. However, tumor-killing T cells in tumor-infiltrating lymphocytes grow slowly in a conventional two-dimensional culture environment, and even die. Moreover, different Expansion efficiency of tumor-killing T cells varies among culture batches
In addition, there are few specific antigens identified for specific tumors, and it is impossible to activate and maintain the lethality of tumor-killing T cells expanded in vitro

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  • Method for amplifying tumor killer T cells in tumor infiltrating lymphocytes

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preparation example Construction

[0036] According to the present disclosure, preferably, the preparation method of the feeder layer material comprises: (1) culturing the 3D tumor organoids in a medium containing microcarriers to obtain a culture medium containing microcarriers coated with 3D tumor organoids (2) Carry out radiation inactivation to the culture medium containing microcarriers coated with 3D tumor organoids, and then separate particles with a particle size greater than 100 μm in the material after radiation inactivation, and use the obtained particles as feeder material.

[0037] Wherein, the medium containing microcarriers contains 5-20 μM Rho kinase inhibitor Y27632, 100-250 ng / mL basic fibroblast growth factor, 50-150 ng / mL epidermal growth factor, 3-10 μg / mL insulin, 30-60% Wnt-3a and 0.2-1.0g / mL bovine serum albumin and other growth factors and RPMI1640 medium.

[0038] According to the present disclosure, preferably, in the culture medium containing microcarriers, the content of microcarrier...

Embodiment

[0042] 1. Preparation of 3D tumor organoids

[0043] 1.1 Microcarrier pretreatment

[0044] Pretreatment was carried out according to the instructions of HyQSpheresCGEN 102-L microcarriers (Thermo Fisher, SV30044). The specific operations included: weighing 5 g of dried microcarriers into siliconized glass bottles, and using 200 ml Mg-free 2+ , Ca 2+ The PBS (200mL per g of microcarrier) was soaked and expanded at room temperature for at least 20 minutes, and the supernatant was discarded; then the microcarrier solution was autoclaved (at a temperature of 121° C., a pressure of 15 psi, and a time of 30 minutes). Discard the liquid after high-temperature sterilization, and store it in a petri dish after siliconization.

[0045] Before using sterile microcarriers, use 200ml Mg-free 2+ , Ca 2+ The supernatant was discarded after rinsing with PBS (200mL per g microcarrier), and the microcarrier was placed in 50ml containing 10μM Rho kinase (ROCK) inhibitor Y27632 (Sigma-Aldric...

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Abstract

The invention relates to a method for amplifying tumor-killing T cells in tumor-infiltrating lymphocytes, and the method comprises the following steps: a, mixing a mononuclear cell suspension with a feeder layer material suspension, and carrying out suspension culture for 96-120 hours to obtain a first culture; wherein the feeder layer material comprises a 3D tumor organ subjected to irradiation inactivation; b, mixing the first culture with a tumor infiltrating lymphocyte culture medium, and carrying out suspension culture for 65-72 hours to obtain a second culture; c, mixing the second culture with the tumor infiltrating lymphocyte culture medium, and carrying out suspension culture for 72-96 hours to obtain a third culture; and d, removing the feeder layer material in the third culture,and separating to obtain the tumor killing T cells. By means of the technical scheme, in-vitro directional amplification of the tumor-killing T cells can be achieved, the amplification efficiency andthe tumor-killing activity of the tumor-killing T cells are greatly improved, and meanwhile the consistency of the amplification efficiency between different amplification batches can be improved.

Description

technical field [0001] The disclosure relates to the technical field of biomedicine, in particular to a method for expanding tumor-killing T cells in tumor-infiltrating lymphocytes. Background technique [0002] Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of lymphocytes present in the tumor stroma, and their common feature is the expression of T cell receptors (TCR). T lymphocytes are the main part, and a small part is MHC non-restricted NK cells. Among them, tumor killer T cells (CD3+CD8+CTL cells) in tumor infiltrating lymphocytes are closely related to the clinical treatment effect of tumors. [0003] At present, the culture of tumor-infiltrating lymphocytes is mainly carried out in a conventional two-dimensional culture environment. However, tumor-killing T cells in tumor-infiltrating lymphocytes grow slowly in a conventional two-dimensional culture environment, and even die. Moreover, different The expansion efficiency of tumor-killing T cells v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/09
CPCC12N5/0636C12N5/0693C12N2500/90C12N2501/2302C12N2513/00C12N2531/00C12N2533/90
Inventor 孙志坚康平李程
Owner 北京科途医学科技有限公司
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