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Method for separating exosomes

A separation method and technology of exosomes, applied in the biological field, can solve problems such as quality reduction, vesicle damage, and time-consuming process, and achieve high yield, reduced centrifugation time, and good results

Inactive Publication Date: 2017-07-21
SHANGHAI KEWEICHUANG BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the process is time-consuming, and repeated centrifugation may damage the vesicles, thereby reducing their quality
In addition, when studying the function of exosomes secreted by specific cells on other cells, it is usually verified by Western blot after a period of infection, which cannot be monitored in real time

Method used

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  • Method for separating exosomes
  • Method for separating exosomes
  • Method for separating exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The construction of embodiment 1 plasmid

[0060] The plasmid pCDNA3.1 was digested with Nhe I endonuclease and restriction endonuclease Hind III, and a band with a vector size of 5.4kb was recovered; the primer CD63-F / R amplified the synthesized CD63 template to obtain a CD63 fragment; because 3flag-F / R are templates for each other, and the 3flag fragment is obtained by PCR amplification. The vector, CD63 fragment and 3flag fragment were added to the HB-infusion seamless cloning kit premix at a molar ratio of 1:3:3, and ligated to obtain the target plasmid pCD63-3*Flag.

[0061] Specific components of the medium in Table 1

[0062]

Embodiment 2

[0063] Example 2 Isolation of exosomes in 293 cells

[0064] 1) Cultivate 293 cells, and when the cell density reaches 70-80%, transfer pCD63-3*Flag into the cells by PEI method, and after culturing for 24 hours, use G418 to screen out the positive clone 293-CD63-3*Flag cells; 293-CD63-3*Flag cells were planted in a 100mm culture dish and cultured for 1 day using the aforementioned cell culture medium;

[0065] 2) Wash the cells with serum-free a-MEM, 5 times, 5ml each time;

[0066] 3) Culture with 10ml 10% a-MEM without exosomes FBS (fetal bovine serum) for 3 days, collect the supernatant, centrifuge at 1000g for 5min, and then centrifuge at 12000g for 30min at 4°C to remove broken cell debris and apoptotic bodies;

[0067] 4) After centrifugation, mix the supernatant with PBS at a volume ratio of 1:1;

[0068] 5) Add flag antibody-coated beads (magnetic beads) to the medium solution containing exosomes, incubate at 4°C for 15 minutes, and the magnetic beads bind to exosom...

Embodiment 3

[0074] Example 3 Isolation of exosomes in Hela cells

[0075] 1) Cultivate Hela cells, and when the cell density reaches 70-80%, transfer pCD63-3*Flag into the cells by PEI method, and after culturing for 24 hours, use G418 to screen out the positive clone Hela-CD63-3*Flag cells; 2 *10^6 Hela-CD63-3*Flag cells were planted in a 100mm culture dish and cultured for 1 day using the aforementioned cell culture medium;

[0076] 2) Wash the cells with serum-free a-MEM, 5 times, 5ml each time;

[0077] 3) Culture with 10ml 10% a-MEM without exosome FBS for 3 days, collect the supernatant, centrifuge at 1000g for 5min, then centrifuge at 12000g at 4 degrees for 30min to remove broken cell debris and apoptotic bodies;

[0078] 4) After centrifugation, mix the supernatant with PBS at a ratio of 1:1;

[0079] 5) Add flag antibody-coated beads (magnetic beads) to the medium solution containing exosomes, incubate at 2°C for 20 minutes, and the magnetic beads bind to exosomes with corresp...

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Abstract

The invention provides a method for separating exosomes. The method at least includes steps of (1), leading CD63 with labels into target cells and culturing the target cells; (2), centrifuging the target cells and then acquiring supernatant; (3), adding magnetic substances into the supernatant and incubating the supernatant and the magnetic substances; (4), arranging the supernatant in magnetic fields; (5), removing the supernatant, adding buffer solution and separation columns into the magnetic substances, eluting the exosomes by the aid of the buffer solution and collecting the exosomes. The magnetic substances contain markers capable of being specifically bound with the labels on the CD63. The exosomes which are about to be eluted are detained on the separation columns. The method for collecting the exosomes has the advantages of high collection efficiency.

Description

technical field [0001] The invention relates to a method for separating exosomes, belonging to the field of biotechnology. Background technique [0002] Exosomes are membranous vesicles with a diameter of about 30-120 nm released into the extracellular matrix after the fusion of intracellular multivesicular bodies (MVBs) and cell membranes. It was first discovered by researchers such as Johnstone when they studied the transformation process of reticulocytes into mature red blood cells. After years of research, scientists have discovered that in addition to reticulocytes, blood cells such as T cells, B cells, platelets, dendritic cells, mast cells, and other non-blood-derived cells such as epithelial cells and tumor cells will secrete exosomes, which are secreted The exosomes will enter into body fluids such as blood, saliva, urine, and milk, and reach other cells and tissues through the circulatory system to produce remote regulation. [0003] Exosomes contain a variety of...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N5/071
CPCC12N5/0018C12N5/0602C12N15/85C12N2500/12C12N2500/14C12N2500/16C12N2500/32C12N2500/34
Inventor 陈意雄朱鹏飞蔡晓龙邹杰
Owner SHANGHAI KEWEICHUANG BIOTECH CO LTD
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