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Method for isolating extracellular vesicle using hydrophobic interaction

A technology for separating cells and vesicles, which can be used in separation methods, cell dissociation methods, general culture methods, etc., and can solve problems such as ineffective removal of lipoproteins

Pending Publication Date: 2020-04-24
ROSETTA EXOSOME CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that lipoproteins cannot be efficiently removed even by density gradient centrifugation, which relies on the density difference between extracellular vesicles and lipoproteins

Method used

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  • Method for isolating extracellular vesicle using hydrophobic interaction
  • Method for isolating extracellular vesicle using hydrophobic interaction
  • Method for isolating extracellular vesicle using hydrophobic interaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Purification and Analysis of Reference Extracellular Vesicles

[0077] Fresh cultures of the colon cancer cell line SW480 were centrifuged at 500 xg for 10 minutes (totally repeated twice). After removing cell debris and precipitates, the supernatant was centrifuged again at 2,000×g for 20 minutes (repeated twice in total) to remove the precipitates.

[0078] A precipitation inducer (8.4% polyethylene glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) was added to the supernatant to first purify and precipitate the extracellular vesicles present therein. After 16 hours in the refrigerator, the supernatant was centrifuged at 12,000 xg for 30 minutes to obtain extracellular vesicles in pellet form. The precipitate was dissolved in HEPES buffer (HEPES buffered saline, 20 mM HEPES, 150 mM NaCl, pH 7.4).

[0079] For a second purification of extracellular vesicles by density and buoyancy, the samples were mixed with OptiPrep TM (30% final concentration) mix and loa...

Embodiment 2

[0083] Example 2: Binding behavior of reference extracellular vesicles to stationary phases with hydrophobic functional groups

[0084] Assays are performed to see if extracellular vesicles bind hydrophobic functional groups. To achieve the above purpose, the HPLC system was used to detect the binding behavior of the reference extracellular vesicles isolated in Example 1.

[0085] First, a column (1 ml) packed with a hydrophobic functional group stationary phase (Phenyl-Sepharose) was washed with 20 column volumes of binding buffer (1M NaCl, 20 mM HEPES, pH 7.2) at a pressure of 1 ml / min. The reference extracellular vesicles isolated in Example 1, according to 4x10 10 Individual amounts were dissolved in the same binding buffer.

[0086] Using the same binding buffer, the reference EV solution was loaded for 5 min onto the washed stationary phase with hydrophobic functional groups, followed by a 5 min wash with the same buffer to remove unbound residues. Then, 20 column vol...

Embodiment 3

[0089] Example 3: Binding behavior of reference extracellular vesicles to stationary phases with hydrophobic functional groups depending on salt concentration

[0090] To examine the binding behavior of reference EVs to stationary phases with hydrophobic functional groups depending on the NaCl concentration, the following experiments were performed.

[0091] Reference extracellular vesicles were purified by polyethylene glycol precipitation. Precipitated reference extracellular vesicles were added to buffers of various NaCl concentrations (20 mM HEPES, pH 7.2 and 0 M, 50 mM, 150 mM, 500 mM NaCl, respectively).

[0092]The empty column was filled with 0.4 ml of a stationary phase (Phenyl-Sepharose) having a hydrophobic functional group, and then washed 3 times with 0.5 ml of water by centrifugation (700×g, 1 minute) to prepare a hydrophobic interaction chromatography column. Each time 0.5ml of the corresponding buffer solution (20mM HEPES, pH7.2 and respectively 0M, 50mM, 150m...

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Abstract

The present invention relates to a method for isolating extracellular vesicles and, more particularly, to a method for isolating extracellular vesicles using hydrophobicity of the extracellular vesicles, and extracellular vesicles isolated using the method. When used, the method for isolating extracellular vesicles according to the present invention allows for isolating, from various animal body fluids including blood and urine or various tissues including cancer tissues, extracellular vesicles free of contamination with lipoproteins that are difficult to eliminate using a conventional method,can solve conventional problems caused by lipoprotein contamination, and is expected to be an essential technology that has a great influence on various research using extracellular vesicles isolatedfrom various animal body fluids or tissues, such as characterization and function research, multi-omics research, excavation of novel biomarkers, diagnosis and treatment, etc.

Description

technical field [0001] The present invention relates to a method for isolating extracellular vesicles, in particular to a method for isolating extracellular vesicles using the hydrophobicity of extracellular vesicles, and to a method for separating extracellular vesicles that do not contain lipoproteins Extracellular vesicles of impurities. Background technique [0002] Extracellular vesicles are nanoscale particles (20-100,000nm) that are naturally released from all cells on Earth and have a cell-derived lipid bilayer membrane structure. In animals, extracellular vesicles have been found in body fluids (e.g., blood, saliva, tears, nasal mucus, sweat, urine, feces, cerebrospinal fluid (CSF), ascites, amniotic fluid, bronchoalveolar lavage fluid, pleural effusion , semen, milk, etc.) and various tumors or normal tissues. [0003] Extracellular vesicles contain various physiologically active substances (such as proteins, lipids, amino acids, RNA, etc.) from secreting cells (...

Claims

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Application Information

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IPC IPC(8): C12N5/00B01D15/36
CPCB01D15/36C12N5/0068C12N5/00C12N2509/00B01D15/20B01D15/327B01J20/267B01J20/287G01N1/34
Inventor 李昌津高用松朴铉泽
Owner ROSETTA EXOSOME CO LTD
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