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Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major

A technology of lymphocytes and cells, which is applied to the preservation of animal cells, vertebrate cells, human or animal bodies, etc. It can solve the problems of multiple blood collection, low purity and low proliferation of activated lymphocytes

Inactive Publication Date: 2012-12-26
JINAN TAISHENG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the disadvantages of low purity and low proliferative ability of the existing activated lymphocyte preparation (mainly referring to CIK cells)
And solve the problem that patients need multiple blood collections due to multiple use of cells
The cryopreservation and resuscitation method of the present invention can solve the problem that patients need multiple blood collections in order to continuously use activated CD3+CD8+-based lymphocytes

Method used

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  • Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major
  • Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major
  • Amplifying, freezing and storing and recovering method of activated lymphocyte with CD3+CD8+as major

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Experimental program
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Embodiment Construction

[0058] 1. Activation of Lymphocytes

[0059] Take 50ml of peripheral blood from the patient, divide it into centrifuge tubes, centrifuge at 1600r / min for 10min, remove the upper layer of plasma, add 30ml of normal saline, mix well, carefully add 30ml of diluted blood cells to the upper layer of 15ml of Ficoll, and then centrifuge for 2000r / min, 20min. After centrifugation, take the middle white blood cell layer, wash it repeatedly with normal saline for 3 times, and add it to the preheated AIM-V medium at 37°C to make the cell concentration 2×10 6 / ml, add anti-CD3 antibody, anti-CD28 antibody and IL-2, so that the concentration of anti-CD3 is 60ng / ml, the concentration of anti-CD28 antibody is 15ng / ml, and the concentration of IL-2 is 4000U / ml. Then put the flask into a carbon dioxide incubator at 37°C, CO 2 The concentration is 5%. From the second day of culture, the cell growth was observed with a microscope.

[0060] 2. Depletion of CD4+CD25+Treg cells

[0061] On th...

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Abstract

The invention discloses culturing, freezing and recovering methods of an activated lymphocyte with CD3+CD8+as major, which can solve problems that a patient needs carrying out blood sampling for many times caused by continuously utilizing the activated lymphocyte with CD3+CD8+as major. The method comprises the following steps of: (1) contacting the extracted lymphocyte of the peripheral blood with IL-2, IL-15, an anti-CD3 antibody and an anti-CD28 antibody, so as to amplify the activated lymphocyte with CD3+CD8+as major; (2) freezing and storing the activated lymphocyte; and (3) recovering the activated lymphocyte. The activated lymphocyte cultured via the method disclosed by the invention has clear components, and comprises few CD4+CD25+Treg cells and more CD8+T lymphocyte; feedback time and frequency of the activated lymphocyte can be adjusted according to other treatments for a patient, such as a radiotherapy or a chemotherapy, so that diseases, such as tumor, infectious diseases and immunodeficiency can be treated well.

Description

technical field [0001] The invention relates to a method for expanding, freezing and resuscitating activated lymphocytes, and belongs to the field of biotechnology. Background technique [0002] The body's immune system has the ability to recognize and kill tumor cells, which has been confirmed by a large number of research data. In recent years, tumor-specific cellular immunotherapy has shown very exciting efficacy in clinical practice. In 2002, RosenbergSA et al. reported in Science magazine the method of adoptive reinfusion of tumor-specific tumor-infiltrating lymphocytes expanded in vitro, and they screened in vitro tumor-infiltrating lymphocytes with the specific killing effect of the patient's own malignant melanocytes ( TILs), a large number of expanded and reinfused to autologous patients. In this method, through the combined action of IL-2 and anti-CD3 antibody in vitro, up to 1011 TILs that are highly specific to tumor cells and specifically recognized by various...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A01N1/02
Inventor 高恒亮郭栋
Owner JINAN TAISHENG BIOLOGICAL TECH CO LTD
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