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A culture method for inducing NK cells in vitro after cryopreserved human pbmc recovery

A technology of NK cells and culture methods, which is applied in the field of in vitro induction of NK cells after recovery from cryopreserved human PBMCs. It can solve the problems of poor viability of NK cells, unstable induction methods, and difficulties in NK cells, and reduce the cost of cell culture. , non-toxic and side effects, and the effect of increasing the amplification factor

Active Publication Date: 2021-11-23
珠海贝索细胞科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this way, the cells after cryopreservation and recovery will be affected by the preparation of the cryopreservation solution and the freezing time. Although there is no obvious difference in morphology, there will be a large loss in number, and it is difficult to induce NK cells.
In addition, the current culture methods of peripheral blood NK cells mainly include factor induction method, trophoblast cell co-cultivation method, and flow sorting cell re-cultivation method; NK cell viability is poor, the induction method is unstable and controversial

Method used

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  • A culture method for inducing NK cells in vitro after cryopreserved human pbmc recovery
  • A culture method for inducing NK cells in vitro after cryopreserved human pbmc recovery
  • A culture method for inducing NK cells in vitro after cryopreserved human pbmc recovery

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Effect test

Embodiment 1

[0061] Example 1 Cell Viability Measurement and Control Experiment

[0062] Collect human peripheral blood from healthy volunteers, separate peripheral blood mononuclear cells, and take out cell samples at the same time point, use the cell recovery method in the culture method for inducing NK cells in vitro after recovery of frozen human PBMCs of the present invention, perform cell recovery, and calculate the number of cells survival rate.

[0063] 1. Preparation of PBMCs

[0064] Carefully pour 20-30ml of blood on 15ml of lymphocyte separation medium; at room temperature, centrifuge at 800xg for 20min, slowly rise and fall, if the blood is stored for more than 2 hours, please increase the centrifugation time to 30min; after centrifugation, the blood is separated 4 layers consisting of plasma (upper layer), mononuclear cell layer between plasma and separation fluid (layer 2), separation fluid (layer 3) and erythrocyte layer (bottom layer); the upper layer of plasma is collect...

Embodiment 2

[0075] Collect 40ml of peripheral blood from healthy volunteers, separate peripheral blood mononuclear cells, and induce NK cells (abbreviated as the experimental group) by using the culture method for inducing NK cells in vitro after recovery of frozen human PBMCs of the present invention, observe the cell morphology at different time points, and take out Count the cell samples, detect the cell phenotype, and observe whether the induced NK cells can meet the requirements of clinical treatment.

[0076] 1. Preparation of PBMCs

[0077] Carefully pour 20-30ml of blood on 15ml of lymphocyte separation medium; at room temperature, centrifuge at 800xg for 20min, slowly rise and fall, if the blood is stored for more than 2 hours, please increase the centrifugation time to 30min; after centrifugation, the blood is separated It consists of 4 layers, consisting of plasma (upper layer), mononuclear cells between plasma and separation fluid (second layer), separation liquid (third layer...

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Abstract

The invention relates to a culture method for inducing NK cells in vitro after cryopreserved human PBMCs are resuscitated. Including three steps of cell recovery, NK cell induction, and NK cell expansion; wherein, in the induction stage of NK cells, use CD 16 and thymosin diluted with PBS solution to coat the culture flask; take serum-free culture medium and add IL‑ 2. IL-12 and IL-15 were prepared as induction medium; serum-free culture solution was added to IL-2 and IL-7 to prepare activation medium; serum-free culture solution was added to IL-2 to prepare expansion medium, On day 3, the half-change medium was used for rehydration for activation culture. Utilizing the culture method of the present invention to induce NK cells in vitro after recovery of cryopreserved human PBMCs is simple and efficient, which not only reduces the cost of cell culture, but also improves the safety of cell culture, and can effectively overcome the difficulty of inducing NK cells from cryopreserved PBMCs and increase the number To achieve the purpose of cell therapy and enhance its anti-tumor, anti-virus and anti-infection capabilities.

Description

technical field [0001] The invention belongs to the technical field of immune cell therapy, and relates to a culture method for inducing NK cells in vitro after resuscitating cryopreserved human PBMC (peripheral blood mononuclear cells). [0002] technical background [0003] Natural killer cells (NK cells), a group of large granular lymphocytes, are the body's first line of defense against pathogens and tumor cells. NK cells are mainly distributed in peripheral blood, accounting for 5-10% of peripheral lymphocytes. NK activity also exists in lymph nodes and bone marrow, but the level is lower compared with peripheral blood. [0004] With the in-depth research on NK cells, NK cells are more and more widely used in the human body. At present, they have good effects in anti-tumor, anti-aging, sub-health treatment, and disease prevention. First, NK cells have cytotoxic function, which can directly kill tumor cells. Secondly, NK cells can bind to receptors on the surface of agi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078C12N5/0783
CPCC12N5/0634C12N5/0646C12N2501/2302C12N2501/2307C12N2501/2312C12N2501/2315C12N2501/599
Inventor 孔伟圣蓝欣何娟娟陈智妍
Owner 珠海贝索细胞科学技术有限公司
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