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Extracellular vesicle separation and enrichment method based on exclusion chromatography and ultrafiltration technology

A technology of size exclusion chromatography and vesicles, which is applied in the field of extracellular vesicle separation and enrichment, can solve the problems of easy clogging of filter membranes, high product purity, and loss of EVs, and achieve the effect of eliminating nucleic acid pollution and improving nucleic acid purity

Active Publication Date: 2019-11-29
北京恩泽康泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Size-exclusion chromatography can separate EVs from miscellaneous proteins according to their particle size, and the product has high purity but low concentration (the number of EVs per unit volume)
Ultrafiltration separates EVs from miscellaneous proteins in body fluids based on the molecular weight cut-off, but complex components in body fluids can easily clog the filter membrane, and when the filter membrane is damaged, a large amount of EVs will be lost

Method used

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  • Extracellular vesicle separation and enrichment method based on exclusion chromatography and ultrafiltration technology
  • Extracellular vesicle separation and enrichment method based on exclusion chromatography and ultrafiltration technology
  • Extracellular vesicle separation and enrichment method based on exclusion chromatography and ultrafiltration technology

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Experimental program
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Effect test

Embodiment 1

[0038] The preparation of embodiment 1 fluorescent liposome

[0039] 1. Preparation of reagent consumables

[0040] Constant pressure control extrusion device (Avestin LiposoFast Basicn) was purchased from Canada Avestin. Phosphatidylcholine, phosphatidylethanolamine, chloroform and methanol were purchased from Sigma-Aldrich. PBS was purchased from Solebol. Fluorescein DHPE was purchased from Thermo Fisher Scientific. Ultrafiltration tubes were purchased from millipore.

[0041] 2. Preparation of liposomes by constant pressure controlled extrusion

[0042] Use a 250 μL sealed glass syringe to add 100 μL chloroform into a 20 mL glass bottle; then use a 100 μL sealed glass syringe to add 30 μL methanol to the same glass bottle; then add 10 mg / mL phosphatidylcholine 216 μL, 10 mg / mL phosphatidylethanolamine 42 μL and 20 μL of 11 mg / mL cholesteryl ester were sequentially added to the above glass bottle; 0.2 mmol of Fluorescein DHPE was added; the organic solvents chloroform a...

Embodiment 2

[0047] Example 2 Purification of EVs by exclusion method based on fluorescent liposome tracking

[0048] 1. Reagent preparation

[0049] Centrifuge commercial human plasma at 1300g for 15min, then transfer the supernatant to a new centrifuge tube and centrifuge at 3000g for 15min. The plasma supernatant was then filtered through a 0.8 μm filter to remove cytoplasmic debris. Take 0.5 mL of plasma supernatant, and add 2 μl of fluorescent liposome solution to it at a volume ratio of 250:1.

[0050] 2. Purification of plasma EVs by size exclusion chromatography

[0051] Pack 5 mL of Sepharose 2B packing into a 6 mL size exclusion column. After fully equilibrating with PBS buffer, add 0.5mL plasma supernatant containing fluorescent liposomes to the exclusion column. Then it was eluted with mobile phase, and 14 fractions were sequentially collected in a volume of 250 μL. The mobile phase used was PBS buffer, and the elution flow rate was about 200-500 μL / min.

[0052] 3. Compo...

Embodiment 3

[0074] Example 3 Comparison of the EVs extraction method based on size exclusion chromatography and ultrafiltration with other methods

[0075] 1. Reagent preparation

[0076] Centrifuge commercial human plasma at 1300g for 15min, then transfer the supernatant to a new centrifuge tube and centrifuge at 3000g for 15min. Then the plasma supernatant was filtered with a 0.8 μm filter membrane to remove the cytoplasm and the residue was set aside.

[0077] 2. EVs extraction by size exclusion chromatography and ultrafiltration (SEC)

[0078] Take three 6ml size exclusion chromatography columns (containing 5mL Sepharose 2B filler). After equilibration, 0.5 mL of filtered plasma supernatant (total 1.5 mL of plasma) was added, and 2 μl of fluorescent liposome solution was added thereto at a volume ratio of 1000:4. Fourteen fractions were collected sequentially in 250 μL volumes. Referring to Experimental Example 2, the corresponding fractions were collected and mixed, then added to...

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Abstract

The invention provides an extracellular vesicle separation and enrichment method based on exclusion chromatography and an ultrafiltration technology. The extracellular vesicle separation and enrichment method comprises the following steps: mixing a fluorescent dye with a body fluid, adding a sample to an exclusion chromatographic column, collecting fractions according to fluorescence intensity, combining the fractions, sequentially treating the combined fractions with protease K and RNase A, and finally carrying out ultrafiltration concentration. The exclusion chromatography and the ultrafiltration method are combined to solve the problem of low product concentration of the exclusion chromatography, and the fluorescent dye is introduced as a tracing external reference, so that the fractionof EVs can be accurately positioned, and the EVs loss caused by damage of a filter membrane can be eliminated before entering the next operation. The invention also provides a kit capable of separating and enriching high-purity extracellular vesicles from body fluid. According to the method, the high-purity EVs can be separated from the body fluid without ultracentrifugation, and meanwhile, in cooperation with protease K and RNase A treatment, nucleic acid pollution from non-vesicle sources in the body fluid can be effectively eliminated, so that the nucleic acid purity of the extracellular vesicles is greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and enriching extracellular vesicles based on size-exclusion chromatography and ultrafiltration technology. Background technique [0002] Cells can secrete vesicles (EVs) with a phospholipid bilayer structure. Because they are rich in nucleic acids, proteins, phospholipids and other biological macromolecules, EVs have broad application prospects in disease diagnosis and treatment. At present, the extraction methods of extracellular vesicles mainly include ultracentrifugation, polymer precipitation, immunocapture, electrostatic adsorption, size-exclusion chromatography, and ultrafiltration. Ultracentrifugation is difficult to meet the needs of clinical testing due to problems such as expensive equipment, cumbersome operation steps, and low recovery rate. The polymer precipitation method is simple to operate and low in cost, but the purity of the product is low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/078C09K11/06
CPCC09K11/06C12N5/0602C12N5/0634C12N2509/00C12N2509/10
Inventor 周卫赵立波孔关义王德键王耀杰闵力
Owner 北京恩泽康泰生物科技有限公司
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