Method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cell

A technology for long-term storage and nucleation of cells, applied in the field of long-term storage and recovery of adult peripheral blood mononuclear cells, it can solve the problems of animal serum, insufficient cell expansion capacity, and low viability of cryopreserved cells, and reduce side effects. Occurrence, effect of ensuring safety and stability

Inactive Publication Date: 2016-02-10
天晴干细胞股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The present invention is proposed to solve the problems existing in the existing technologies such as the low extraction rate of PBMC, the cell freezing solution containing animal serum, the low survival rate of frozen cells, and the insufficient cell expansion ability after resuscitation.

Method used

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  • Method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cell
  • Method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cell
  • Method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cell

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specific Embodiment approach 1

[0024] Embodiment 1: A method for long-term storage and recovery of adult peripheral blood mononuclear cells in this embodiment is carried out according to the following steps:

[0025] 1. Separation and purification of collected adult peripheral venous blood to obtain peripheral blood mononuclear cells;

[0026] 2. Washing and purifying the obtained peripheral blood mononuclear cells;

[0027] 3. Dilute the peripheral blood mononuclear cells by resuspending them in the cryopreservation medium, so that the density of the cell suspension reaches 1~10×10 7 a / mL;

[0028] 4. Divide the diluted cell suspension into 1.8mL cryopreservation tubes, add 1mL to each tube, and keep the sample for inspection, put the cell cryopreservation tubes into a 4°C pre-warmed program cooling box, and transfer to - 80 ℃ refrigerator for temporary storage;

[0029] 5. The reserved frozen cell suspension is tested for indicators such as bacteria, fungi, endotoxin and mycoplasma. After the indicator...

specific Embodiment approach 2

[0033] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that the method for obtaining peripheral blood mononuclear cells in step 1 is: centrifuge to collect adult peripheral venous blood, inactivate it in a water bath, and centrifuge to collect the plasma supernatant. Aliquot one 10mL tube and store at -80°C for later use; use lymphocyte separation medium to separate mononuclear cells from blood cells, and collect the mononuclear cells. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0034] Embodiment 3: The difference between this embodiment and Embodiment 1 is that the obtained peripheral blood mononuclear cells are cleaned in step 2, and the specific operation of purification is as follows: wash the mononuclear cells twice with compound electrolyte solution, collect the final The supernatant was washed once, and tested for bacteria, fungi, mycoplasma and endotoxin. After passing the test, the cells collected by centrifugation were set aside. Others are the same as in the first embodiment.

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Abstract

The invention discloses a method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cells, and relates to a separation, cryopreservation and resuscitation method of adult peripheral blood mononuclear cells. The method comprises the following steps: I, collecting peripheral venous blood of a healthy adult by using a blood taking bag, and separating and purifying the mononuclear cells in the blood; II, adding related cryopreservation reagents necessary for cryopreservating cells so as to cryopreservate the separated mononuclear cells; III, randomly sampling, and detecting bacteria, fungi, endotoxin and mycoplasma in a cell cryopreservation mixed liquid; and IV, resuscitating the cells, suspending in specially prepared cell resuscitation lotion, performing centrifugal washing twice, and further culturing. As the peripheral blood mononuclear cells used in the method are taken from a patient self, the related reagents used in the test are free of exogenous material, related ethical issues can be avoided, and immunological rejection can be avoided after the cells are injected back to the body. The method aims to store healthy adult immune cells for later treatment on diseases related to tumor.

Description

technical field [0001] The present invention relates to a method for long-term storage, resuscitation and culture of adult peripheral blood mononuclear cells, in particular to a method for efficient expansion of cells after cryopreservation and resuscitation, which can be used for clinical treatment of adult immune cells storage and later resuscitation culture project. Background technique [0002] Immune cell (immune cell) is a common name for white blood cells, including lymphocytes and various phagocytes, etc., and also specifically refers to lymphocytes that can recognize antigens and generate specific immune responses. Lymphocytes are the basic components of the immune system and are widely distributed in the body. Mainly T lymphocytes and B lymphocytes are activated by antigen stimulation, divide and proliferate, and produce specific immune responses. T lymphocytes are a multifunctional cell population. In addition to T lymphocytes and B lymphocytes, there is another...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078A01N1/02
Inventor 张怡兰凤翔李琳刘艳青
Owner 天晴干细胞股份有限公司
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