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Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes

A technology of lymphocyte and tumor infiltration, applied in the field of cell culture, can solve the problems of high irradiation cost and unfavorable wide use, and achieve the effect of expanding the scope of application, promoting the proliferation of TIL, and achieving good effect.

Inactive Publication Date: 2020-01-03
SOUTHWEST MEDICAL UNIVERISTY
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a low-cost preparation of feeder cells for the rapid cultivation of tumor-infiltrating lymphocytes in view of the problems in the prior art that irradiating PBMC with γ-rays to prepare feeder cells has high irradiation costs and is not conducive to widespread use method

Method used

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  • Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes
  • Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes
  • Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes

Examples

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Embodiment

[0038] A preferred embodiment of the present invention provides a method for preparing feeder cells for rapid culture of tumor-infiltrating lymphocytes. The specific steps are as follows:

[0039] 1. Preparation of feeder cells:

[0040]1) Isolation and culture of lymphocytes from the patient's autologous peripheral blood: After extracting 50 mL of the patient's autologous peripheral blood, dilute it with phosphate buffer saline at a ratio of 1:1, then add lymphocyte separation medium to separate lymphocytes, and then use serum-free X-VIVO medium (adding 40ng / mL of IL-2 and 200ng / mL of CD3 / CD28 antibody) in 5%, CO 2 , 37°C incubator for cultivation.

[0041] 2) Preparation of feeder cells: After the isolated lymphocytes were cultured overnight, the cells were counted, and the cell density was adjusted to 1.5×10 6 cells / mL, add mitomycin C (final concentration: 50 μg / mL) to treat for 1.5 hours, centrifuge at 1000rpm / min for 5min, discard the supernatant, then resuspend and wa...

experiment example 1

[0047] The feeder cells produced by γ-ray irradiation and the method of the embodiment of the present invention were used to prepare the feeder cells respectively, and the feeder cells prepared by the two methods were co-cultured with non-small cell lung cancer TIL respectively, and the results were as follows figure 1 shown.

[0048] It can be seen from the figure that the difference in the proliferation efficiency of the cells is small, and the growth factor of the cells is higher after the feeder cells prepared by the method of the embodiment of the present invention are co-cultured with the non-small cell lung cancer TIL for 21 days.

experiment example 2

[0050] Feeder cells produced by γ-ray irradiation and the method of the embodiment of the present invention were used to prepare feeder cells respectively, and flow cytometry was used to detect the content of killer T cells (CD3+CD8+) after the feeder cells prepared by the two methods were co-cultured with TIL for 14 days , the result is as figure 2 and 3 shown; the results show similar effects.

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Abstract

The invention discloses a preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes. The preparation method comprises the steps that peripheral blood lymphocytes are treated by using mitomycin C to obtain the feeder cells, and then the feeder cells and pre-cultured tumor infiltrating lymphocytes are co-cultured. The efficiency of expanding the tumor infiltrating lymphocytes of the feeder cells produced by the preparation method is better than that of the feeder cells produced by gamma-ray irradiation, and meanwhile the cost of using the preparation method is significantly lowered. The preparation method is simple and easy to operate, and can be widely applied and promoted.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for preparing feeder cells for rapid culture of tumor-infiltrating lymphocytes. Background technique [0002] Tumor infiltrating lymphocytes (Tumor infiltrated lymphocytes, TIL) are lymphocytes that leave the blood circulation and migrate to the vicinity of the tumor, including T cells, B cells, NK cells, etc. They can play a role in killing cancer cells. The amount of TIL in a tumor is an important indicator to predict the prognosis of cancer patients and the response to immunotherapy. TIL therapy is mainly to rapidly amplify TIL isolated from fresh tumor tissue of the patient in an in vitro cell culture environment, expand the number of TIL to the amount that can be reinfused to the patient, and then infuse it back into the patient. [0003] The currently known technology of TIL in vitro expansion uses the isolated TIL to be pre-cultured in vitro for...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N15/01
CPCC12N5/0636C12N15/01C12N2502/1114
Inventor 肖占刚赵曰水俞静文庆莲李静
Owner SOUTHWEST MEDICAL UNIVERISTY
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