Exosome enrichment method

An exosome and enrichment technology, applied in the preparation of test samples, can solve problems such as high recovery efficiency, and achieve the effect of reducing processing time and fast processing

Active Publication Date: 2019-11-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method typically requires overnight incubation to achieve high recovery efficiencies

Method used

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Experimental program
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Effect test

Embodiment 1

[0018] Centrifuge 10 mL of human urine at 200 g for 5 minutes, put the supernatant in a new centrifuge tube, centrifuge at 2000 g for 20 minutes, and filter through a 0.22 micron membrane filter. Add the PBS solution that mass concentration is 50% PEG10000 in filtrate, make PEG final mass concentration be 8%, add sodium chloride and make final concentration be 150mM, add DMSO and make final volume concentration be 5%, sample is placed in -20°C, after standing for 30 minutes, put the sample at 4°C to melt. The melted samples were centrifuged at 500 g for 30 min. The pellet obtained after centrifugation was the exosome sample. NTA analysis and electron microscopy results showed enriched exosomes. The results of proteomics showed that the technical reproducibility of the sample processing flow was high (attached figure 1 ).

Embodiment 2

[0020] Centrifuge 10 mL of HELA cell culture solution at 500 g for 5 minutes, centrifuge the supernatant at 4000 g for 20 minutes, and filter through a 0.22-micron membrane filter. Add 50% PEG aqueous solution to the filtrate to make the final mass concentration of PEG20000 8%, add DMSO to make the final volume concentration 10%, place the sample at -80°C, and after standing for 10 minutes, place the sample Thawing was performed at 37°C. The thawed sample was centrifuged at 5000 g for 5 min. The pellet obtained after centrifugation was the exosome sample. NTA analysis and electron microscopy results showed enriched exosomes. Proteomics results showed that there were more exosomal proteins.

Embodiment 3

[0022] Dilute 0.2 mL of human plasma to 10 mL with PBS, centrifuge at 500 g for 5 minutes, put the supernatant in a new centrifuge tube, centrifuge at 2000 g for 20 minutes, and centrifuge at 10000 g for 1 h. Add the PBS solution that mass concentration is 50% PEG2000 in filtrate and make PEG final mass concentration be 12%, add sodium chloride and make final concentration be 50mM, add butanol and make final volume concentration be 5%, sample is placed in After standing at -60°C for 20 minutes, the sample was melted at 37°C. The melted samples were centrifuged at 4000g for 20min. The pellet obtained after centrifugation was the exosome sample. NTA analysis and electron microscopy results showed enriched exosomes. Proteomics results showed that most of the proteins belonged to the Exocart database.

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Abstract

The invention relates to an exosome enrichment method and an application of the exosome enrichment method in exosome fast enrichment concentration in a biological sample. The method comprises the following steps of carrying out differential centrifugation treatment or ultrafiltration treatment on a biological sample to remove cell interference in the sample; adding a PEG solution into a supernate,and standing for a period of time at a low temperature; and putting the sample at a proper temperature to form a uniform solution, carrying out centrifuging at a low speed, and obtaining precipitatewhich is the exosome.

Description

technical field [0001] The invention relates to a method for enriching exosomes and its application in the rapid enrichment of exosomes in biological samples. Specifically, it is a method based on PEG polymer capturing exosomes under low temperature conditions, and then enriching exosomes under low-speed centrifugation conditions. Background technique [0002] In the 1960s and 1970s, researchers found many membrane-coated vesicles outside cells, such as cartilage, blood and cell culture fluid (J. Cell Biol. 1969, 41, 59-72. ). Initially, it was discovered that some of these vesicles were released by budding out of the plasma membrane. Until the 1980s, a more complex method of vesicle secretion was discovered. The endosomes in the cell first form multivesicular bodies, and release the vesicles contained therein to the outside of the cell through plasma membrane fusion (J. Biol. Chem. 1987, 262, 9412-9420). The secreted form of such vesicles has been studied in detail, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/40
CPCG01N1/40
Inventor 张丽华随志刚杨开广侯瑞袁辉明张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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