Efficient PD-1-CD8+T cell culture method

A PD-1-CD8, culture method technology, applied in the field of PD-1-CD8+ T cell culture, can solve the problems of poor clinical treatment effect, low efficiency, microbial contamination amplification efficiency, etc., to reduce the probability of microbial contamination , high cell expansion efficiency, and the effect of improving clinical efficacy

Inactive Publication Date: 2017-04-26
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the defects of poor clinical treatment effect, susceptibility to microbial contaminatio

Method used

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  • Efficient PD-1-CD8+T cell culture method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] A high-efficiency method for culturing PD-1-CD8+ T cells, the specific steps are:

[0064] Step 1. Obtain TIL cells:

[0065] (1) Extract TIL cells:

[0066] a. Take fresh tumor tissue from the patient, and under aseptic conditions, remove the yellow fat tissue and blood clot around the tumor, leaving the central part for later use;

[0067] b. Under a sterile environment, chop the lump treated in step a into 1.5mm 3 A small piece, placed in a volume of 40ml, with a bottom area of ​​10 cm 2 In the G-Rex10 breathable bottle;

[0068] c. Add 25ml of digestive solution to the G-Rex10 gas bottle, shake it for 1min, and place the G-Rex10 gas bottle at 37°C, 5% CO 2 In the incubator, incubate for 30min;

[0069] d. Repeat step c twice until the mass is completely digested to obtain a suspension;

[0070] e, the cell suspension prepared in step d is further separated and purified by density gradient centrifugation using Ficoll;

[0071] f. Put all cell suspensions in a ...

Embodiment 2

[0089] The invention discloses a highly efficient method for culturing PD-1-CD8+ T cells. The specific steps are:

[0090] Step 1. Obtain TIL cells:

[0091] (1) Extract TIL cells:

[0092] a. Take fresh tumor tissue from the patient, and under aseptic conditions, remove the yellow fat tissue and blood clot around the tumor, leaving the central part for later use;

[0093] b. Under a sterile environment, chop the lump treated in step a into 1mm 3 A small piece, placed in a volume of 40ml, with a bottom area of ​​10 cm 2 In the G-Rex10 breathable bottle;

[0094] c. Add 10ml of digestive solution to the G-Rex10 gas bottle, shake it for 1min, and place the G-Rex10 gas bottle at 37°C, 5% CO 2 In the incubator, incubate for 30min;

[0095] d. Repeat step c twice until the mass is completely digested to obtain a suspension;

[0096] e, the cell suspension prepared in step d is further separated and purified by density gradient centrifugation using Ficoll;

[0097] f. Put all...

Embodiment 3

[0115] The invention discloses a highly efficient method for culturing PD-1-CD8+ T cells. The specific steps are:

[0116] Step 1. Obtain TIL cells:

[0117] (1) Extract TIL cells:

[0118] a. Take fresh tumor tissue from the patient, and under aseptic conditions, remove the yellow fat tissue and blood clot around the tumor, leaving the central part for later use;

[0119] b. Under a sterile environment, chop the lump treated in step a into 2mm 3 A small piece, placed in a volume of 40ml, with a bottom area of ​​10 cm 2 In the G-Rex10 breathable bottle;

[0120] c. Add 40ml of digestive solution to the G-Rex10 gas bottle, shake it for 1min, and place the G-Rex10 gas bottle at 37°C, 5% CO 2 In the incubator, incubate for 30min;

[0121] d. Repeat step c twice until the mass is completely digested to obtain a suspension;

[0122] e, the cell suspension prepared in step d is further separated and purified by density gradient centrifugation using Ficoll;

[0123] f. Put all...

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Abstract

The invention discloses an efficient PD-1-CD8+T cell culture method, which specifically comprises: extracting TIL cells; screening the TIL cell suspension by using a flow cell sorter to obtain purified PD-1-CD8+T cells; adding the purified PD-1-CD8+T cells to a G-Rex gas permeable bottle, and culturing for 1 week with a complete culture medium; and rapidly amplifying the PD-1-CD8+T cells. The efficient PD-1-CD8+T cell culture method of the present invention has advantages of effective microbial contamination rate reducing, high amplification efficiency, high cytotoxic activity, and the like.

Description

technical field [0001] The present invention relates to the field of tumor adoptive immune cell therapy, and specifically relates to an efficient method for culturing PD-1-CD8+ T cells. Background technique [0002] TIL cell therapy is a kind of immunotherapy, which is gradually developing in recent years. Its original proposer is Rosenberg. TIL (Tumor infiltrating lymphocytes) refers to tumor-infiltrating lymphocytes, in which the main effector cells are CD8+ T cells. In the tumor tissue, most of them are tumor cells, and there are also a small number of lymphocytes. Some of these lymphocytes are T cells targeting tumor-specific mutant antigens, which can kill tumor cells. However, due to some reasons, such as the tumor microenvironment and PD-1, their functions are inhibited, and they cannot effectively kill tumor cells in tumor tissues. However, scientists enrich certain types of lymphocytes in these tumor tissues through some in vitro culture methods, and then reinfuse t...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2500/84C12N2501/2302C12N2509/00
Inventor 韦玉军陆宝石李航
Owner 安徽安龙基因科技有限公司
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