Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Exosome purification method

A purification method and exosome technology, applied in biochemical equipment and methods, cell dissociation methods, microorganisms, etc., can solve problems such as difficult removal, high osmotic pressure, and time-consuming density gradient balance, and achieve the effect of improving purity

Inactive Publication Date: 2021-09-17
微纳核酸生物医药广东有限公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using sucrose density gradient and cesium chloride density gradient centrifugation to achieve density gradient equilibrium is time-consuming and difficult to remove. Sucrose solution and cesium chloride solution tend to form high osmotic pressure, which is not conducive to maintaining the integrity of exosomes.

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
  • Exosome purification method
  • Exosome purification method
  • Exosome purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] As a method for purifying exosomes according to an embodiment of the present invention, the method includes the following steps:

[0025] (1) Centrifuge 50 mL of exosome-rich cell culture medium at 4°C and 10,000 g for 30 min;

[0026] (2) Transfer the supernatant obtained in step (1) to an ultracentrifuge tube (BECKMAN, Cat#: 326823), and add 100 μL of 40% (w / v) iopamidol solution to the bottom of the ultracentrifuge tube; centrifuge at 100,000 g 70min, to obtain the exosome crude extract, during centrifugation, the volume ratio of the supernatant obtained in step (1) and iopamidol is 500:1;

[0027] (3) Dilute 1mL of crude exosome extract with PBS buffer solution to 5mL, mix well, transfer to a new ultracentrifuge tube (BECKMAN, Cat#: 326819), add 500uL exosome pure ( thawing after freezing), centrifuged at 100000g for 70min; the exocrine pure is 25% (w / v) iopamidol solution;

[0028] (4) Collect in layers from the bottom, 100 μL of liquid in each layer, from the bo...

Embodiment 2

[0031] As a method for purifying exosomes according to an embodiment of the present invention, the method includes the following steps:

[0032] (1) Centrifuge 50mL of exosome-rich cell culture medium at 4°C and 9000g for 25min;

[0033] (2) Transfer the supernatant obtained in step (1) to an ultracentrifuge tube (BECKMAN, Cat#: 326823), and add 100 μL of 42% (w / v) iopamidol solution to the bottom of the ultracentrifuge tube; centrifuge at 110,000 g 65min, the exosome crude extract was obtained, and during centrifugation, the volume ratio of the supernatant obtained in step (1) to iopamidol was 550:1;

[0034] (3) Dilute 1mL of crude exosome extract with PBS buffer solution to 5mL, mix well, transfer to a new ultracentrifuge tube (BECKMAN, Cat#: 326819), add 500uL exosome pure ( thawing after freezing), centrifuged at 100000g for 65min; the exocrine pure is 28% (w / v) iopamidol solution;

[0035] (4) Collect in layers from the bottom, 100 μL of liquid in each layer, from the ...

Embodiment 3

[0037] As a method for purifying exosomes according to an embodiment of the present invention, the method includes the following steps:

[0038] (1) Centrifuge 50 mL of exosome-rich cell culture medium at 4°C and 11,000 g for 35 min;

[0039] (2) Transfer the supernatant obtained in step (1) to an ultracentrifuge tube (BECKMAN, Cat#: 326823), and add 100 μL of 40% (w / v) iopamidol solution to the bottom of the ultracentrifuge tube; centrifuge at 90,000 g 75min, to obtain the exosome crude extract, during centrifugation, the volume ratio of the supernatant obtained in step (1) and iopamidol is 600:1;

[0040] (3) Dilute 1mL of crude exosome extract with PBS buffer solution to 5mL, mix well, transfer to a new ultracentrifuge tube (BECKMAN, Cat#: 326819), add 600uL exosome pure ( thawing after freezing), centrifuged at 110000g for 75min; the exocrine pure is 30% (w / v) iopamidol solution;

[0041] (4) Collect in layers from the bottom, 100 μL of liquid in each layer, from the bot...

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 provides an exosome purification method. The exosome purification method comprises the following steps: (1) centrifuging a cell culture solution rich in exosomes; (2) centrifuging supernate obtained in the step (1) and a 40% (w / v) iopamidol solution together to obtain an exosome crude extract, wherein the volume ratio of the supernate obtained in the step (1) to iopamidol is (500-600):1; (3) diluting the exosome crude extract by 4-6 times with a buffer solution, and performing centrifuging together with the exosomes, wherein the exosomes are located at the lower part of the exosome crude extract; the volume ratio of the exosome crude extract to the exosome is 2:(1-1.5); and the exosome is a 25-30% (w / v) iopamidol solution; and (4) respectively collecting products obtained after centrifugal layering in the step (3). According to the purification method disclosed by the invention, the iopamidol with a certain concentration is subjected to ultracentrifugation, so that a continuous density gradient can be quickly formed; and the purification method can be used for separating the exosome with relatively high purity and is beneficial to improving the purity of the obtained exosome.

Description

technical field [0001] The invention relates to the field of exosome preparation, in particular to a method for purifying exosomes. Background technique [0002] Use a certain medium to form a continuous or discontinuous density gradient in the centrifuge tube, place the cell suspension or homogenate on the top of the medium, and separate the cells or different components in the cells through the action of gravity or centrifugal force field the goal of. The commonly used density gradient media are mainly sucrose and cesium chloride. Exosomes are nano-sized lipid vesicles formed by the fusion of plasma membranes to form multivesicular bodies through internal invagination and then released to the outside of the cell. DNA, RNA and most proteins are light. Traditional differential ultracentrifugation is one of the most commonly used methods for exosome isolation and purification, but when exosomes are centrifuged to the bottom of the centrifuge tube, the integrity of exosomes...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00
CPCC12N5/00C12N2509/10
Inventor 方杨武王奕李东升于振亚
Owner 微纳核酸生物医药广东有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com