Culture method of high-memory phenotype tumor-infiltrating T lymphocytes

A technology of tumor infiltration and lymphocytes, which is applied in the field of cell culture, can solve the problems of low proportion of memory lymphocytes, complicated operation procedures, and low activity, and achieve high-efficiency proliferation activity, high-efficiency method, and high proportion

Active Publication Date: 2021-05-11
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first object of the present invention is to provide a method for culturing tumor-infiltrating T lymphocytes with a high-memory phenotype, which solves the problems of complicated operating procedures, long time-consuming, low activity, and relatively high proportion of memory lymphocytes in existing TIL cell expansion methods. low level problem

Method used

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  • Culture method of high-memory phenotype tumor-infiltrating T lymphocytes
  • Culture method of high-memory phenotype tumor-infiltrating T lymphocytes
  • Culture method of high-memory phenotype tumor-infiltrating T lymphocytes

Examples

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Embodiment 1

[0036] Example 1 High memory phenotype tumor infiltrating T lymphocyte culture

[0037] To cultivate high-memory phenotype tumor-infiltrating T lymphocytes, the steps are as follows: Aseptically excise tumor tissue from patients with advanced melanoma, and the tumor size is ≥ 1cm 3 , placed in the culture medium, mechanically shredding and digesting the above-mentioned tumor body under aseptic and cold-chain conditions to obtain a single-cell suspension, separate and extract tumor-infiltrating T lymphocytes, and add them to the culture medium containing the separation-inducing culture medium After 14 days of culture, a cell suspension of tumor-infiltrating T lymphocytes with a high memory phenotype was obtained. The separation and induction culture medium contained IL-2, IL-7, and IL-15 at a concentration of 10 ng / ml, respectively. , 10ng / ml, and 20ng / ml medium, you can prepare 10ug / ml, 10ug / ml, and 20ug / ml of IL-2, IL-7, and IL-15 respectively, and then dilute them to the abo...

Embodiment 2

[0040] Example 2 Cell experiment

[0041] The high-memory phenotype TILs cells obtained in Example 1 were co-cultured with tumor cells in vitro to simulate the killing ability of TILs on tumor cells in vivo. After 4-6 hours of co-culture in vitro by CFSE-PI double-staining labeling method, flow cytometry Detecting the proportion of tumor cells with PI+, it was found that the tumor cells had undergone significant apoptosis, and the killing rate was as high as 67.8%, which was 15% higher than that of traditional TILs, suggesting that TILs with high memory phenotype had a strong tumor killing ability ( Figure 4 ).

Embodiment 3

[0042] Embodiment 3 animal experiments

[0043] A subcutaneous xenograft tumor model was constructed in immunodeficient mice, and TILs were injected into the tail vein to observe the safety, tolerance, and tumor progression of the mice. It was found that the body weight of the mice injected with TILs had no significant change compared with the control group ( Figure 5 A), suggesting that the TILs product is well tolerated; at the same time, it can be observed that the subcutaneous tumors of mice injected with TILs are significantly reduced ( Figure 5 B).

[0044] In addition, the high-memory phenotype TILs cell product obtained in Example 1 was cryopreserved in liquid nitrogen, and after recovery, the activity of the cell preparation was still maintained at more than 90%, which had good stability ( Image 6 ).

[0045] Different batches of high-memory phenotype tumor-infiltrating T lymphocytes were prepared according to the steps in Example 1, and the detection results of ...

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Abstract

The invention discloses a culture method of high-memory phenotype tumor-infiltrating T lymphocytes. The culture method comprises the following steps of aseptically cutting tumor tissues and putting the tumor tissues into a culture solution, mechanically cutting and digesting tumor bodies under an aseptic cold chain condition to obtain a single-cell suspension, separating and extracting the tumor-infiltrating T lymphocytes, and obtaining the high-memory phenotype tumor-infiltrating T lymphocytes by adding the tumor-infiltrating T lymphocytes into a culture medium containing a separation induction culture solution for in-vitro amplification and induced differentiation, and culturing for 7-21 days, wherein the separation induction culture solution is a culture medium containing IL-2, IL-7 and IL-15 with the concentrations of 10ng / ml, 10ng / ml and 20ng / ml respectively. According to the method, the TIL cells are subjected to in-vitro amplification and induced differentiation by separating and inducing the culture solution, the high-memory phenotype TILs with efficient proliferation activity and strong tumor killing ability are obtained to the maximum extent, the method is simple and efficient, the proportion of the memory phenotype T lymphocytes is high, and the TILs can be used for treating advanced malignant melanoma.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing tumor-infiltrating T lymphocytes (TILs) with a high-memory phenotype. Background technique [0002] Malignant melanoma (melanoma for short) is the most malignant skin tumor, and its incidence has been increasing at an annual rate of 3%–5% in recent years. Surgery can remove early-stage melanoma, but the effective rate of conventional treatment after distant metastasis of melanoma is low. The 5-year survival rate is only 10%-20%, and the median survival time is only 6-9 months. The prognosis is extremely poor. Melanoma has a very high mutation load and immunogenicity. Immunotherapy is particularly effective in melanoma. However, more than 50% of melanoma patients still do not respond to immunotherapy, and some patients have explosive tumor progression. Therefore, optimizing the immunotherapy It is of great clinical significance to explore treatment met...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0636A61K35/17A61P35/00C12N2509/10C12N2509/00C12N2501/2302C12N2501/2307C12N2501/2315
Inventor 顾建英周宇红卢莉莉孙磊吴伟忠卫传元高子煦肖淑秀宋正清白金金
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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