Multi-stage purification method and apparatus of extracellular vesicles using aqueous solution two-phase system

Patent metadata

Field	Value
Publication number	US-2018105778-A1
Application number	US-201615560561-A
Country	US
Kind code	A1
Filing date	Mar 3, 2016
Priority date	Mar 24, 2015
Publication date	Apr 19, 2018
Grant date	—

Abstract

This disclosure relates to a multi-stage method and apparatus for purifying extracellular vesicles using an aqueous two-phase system, in which extracellular vesicles mixed and contaminated with proteins can be isolated and purified in a large amount at high purity within a short time through a multi-stage purification process using an aqueous two-phase system, thereby removing 95% or more of proteins and obtaining high-purity extracellular vesicles, resulting in very high processing efficiency compared to conventional techniques. In particular, the method of the disclosure does not require an expensive device or material such as an ultracentrifuge or an antibody, and can be performed at low cost and is thus economical and highly competitive. Furthermore, the extracellular vesicles thus isolated and purified can be employed in analysis methods such as RT-PCR or western blot, and can be utilized for research fields and disease diagnosis using the same.

First claim

1 - 18 . (canceled)

19 . A multi-stage purification method of extracellular vesicles using an aqueous two-phase system, comprising the steps of:
(a) preparing a mixture by mixing a first material and a second material with a body fluid containing proteins or an aqueous solution containing extracellular vesicles mixed and contaminated with proteins; (b) subjecting the mixture to phase separation into an upper solution and a lower solution to give an aqueous two-phase system; (c) removing the upper solution including the first material from the aqueous two-phase system; (d) supplying and mixing the remaining lower solution with a new upper solution including the first material; and (e) performing the steps (b) to (d) at least two times and then recovering the extracellular vesicles from the lower solution, wherein the body fluid is at least one selected from the group consisting of whole blood, serum, peritoneal fluid, breast milk, and urine.

20 . The multi-stage purification method of claim 19 , wherein the first material is selected from among water, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, and ficoll, and
the second material is selected from among EOPO (ethylene oxide propylene oxide), dextran, levan, poly(vinyl methyl ethyl ether), ammonium sulfate, sodium sulfate, magnesium sulfate, potassium phosphate, and sodium carbonate.

21 . The multi-stage purification method of claim 20 , wherein the first material is water and the second material is EOPO.

22 . The multi-stage purification method of claim 20 , wherein the first material is polyethylene glycol and the second material is selected from among dextran, levan, poly(vinyl methyl ethyl ether), ammonium sulfate, sodium sulfate, magnesium sulfate, potassium phosphate, and sodium carbonate.

23 . The multi-stage purification method of claim 20 , wherein the first material is selected from among polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, and ficoll, and the second material is dextran.

24 . The multi-stage purification method of claim 20 , wherein the first material is polyethylene glycol and the second material is dextran.

25 . The multi-stage purification method of claim 19 , further comprising controlling an attractive force or a repulsive force of molecules in the aqueous two-phase system by adding an additive to the aqueous two-phase system, after the step (a) and before the step (b).

26 . The multi-stage purification method of claim 19 , wherein the phase separation in the step (b) is performed through centrifugation at 500˜2,000×g-force.

27 . The multi-stage purification method of claim 19 , wherein the step (b) further comprises applying ultrasonic waves or supplying microbubbles to the aqueous two-phase system.

28 . A method of performing an assay through ELISA, RT-PCR, western blot, proteomics or genomics using extracellular vesicles isolated and purified by the multi-stage purification method of claim 19 .

Assignees

• Postech Acad Ind Found

Inventors

• Park Jae Sung
• Shin Hyun Woo

Classifications

• B01D11/0488

  Flow sheets · CPC title

• C12M1/26

  Inoculator or sampler · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01D11/0492

  Applications, solvents used · CPC title

• B01D17/0217

  by centrifugal force · CPC title