Culture method for in-vitro induction of NK cell after recovery of cryopreserved human PBMC

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 unstable induction methods, poor NK cell viability, and NK cell difficulties, and improve cell expansion. Multiples, no toxic side effects, and the effect of improving induction efficiency

Active Publication Date: 2020-09-29
珠海贝索细胞科学技术有限公司
View PDF5 Cites 5 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Culture method for in-vitro induction of NK cell after recovery of cryopreserved human PBMC
  • Culture method for in-vitro induction of NK cell after recovery of cryopreserved human PBMC
  • Culture method for in-vitro induction of NK cell after recovery of cryopreserved human PBMC

Examples

Experimental program
Comparison scheme
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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a culture method for in-vitro induction of an NK cell after recovery of a cryopreserved human PBMC. The culture method comprises three steps including cell recovery, inductionof the NK cell and NK cell proliferation, wherein in the stage of indication of the NK cell, CD 16 and thymosin are diluted with a PBS solution for culture flask enveloping; IL-2, IL-12 and IL-15 areadded to a serum-free culture solution for preparation of an induction medium; IL-2 and IL-7 are added to the serum-free culture solution for preparation of an activation culture medium; and IL-2 isadded to the serum-free culture solution for preparation of a proliferation culture medium, and half-liquid replacement is carried out on the day 3 for solution replacement and activation culture. Themethod for in-vitro induction of the NK cell after recovery of the cryopreserved human PBMC is simple and efficient, reduces the cost of cell culture and also increases safety of cell culture, can effectively overcome the difficulty in inducing the NK cell with the cryopreserved PBMC and deficiency in a proliferation amount, reaches a purpose of cell treatment and enhances resistance to tumors, viruses and infection thereof.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products