Method for separating and purifying exosomes

An exosome and purification column technology, applied in the biological field, can solve the problems of slow speed and low purification degree, and achieve the effect of simple operation, fast purification speed and accelerated purification degree.

Pending Publication Date: 2021-04-09
中聚生物技术(广东)集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Exosomes are 30-200nm-diameter encapsulated cystic vesicles with double-layered lipid molecules, which are selectively packaged and released by living cells, and are abundant in human body fluids. Exosomes contain different types of Lipids, nucleic acids, and proteins, etc., related studies have also shown that exosomes play a huge role in intercellular communication and physiological and pathological processes; at present, the removal of foreign proteins in exosomes is an urgent solution However, in the existing separation and purification methods, the exosome suspension still contains a large amount of impurity proteins, which are not pure exosomes, and the purification degree is low and the rate is slow.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] A method for isolating and purifying exosomes, specifically according to the following steps:

[0019] S1: Material preparation, 30ml of biological fluid, 1 differential centrifuge tube, 1 purification column, 1 affinity chromatography column, 5g of hydroxyapatite, 20ml of sodium dihydrogen phosphate solution, 10ml of lectin, 40ml of sucrose solution, Phosphate buffered saline solution, 1 storage tank, 1 magnetic stirrer, 1 sterile filter;

[0020] S2: For the initial separation, put the biological fluid into a differential centrifuge tube, and centrifuge at 300g for 3min, 3000g for 3min, and 10000g for 3min to remove cells, dead cells and cell debris respectively; The crude extract of exosomes is centrifuged at 100,000 g for 3 minutes to remove contaminating proteins to obtain exosome suspension; the exosomes are separated by differential centrifugation;

[0021] S3: For the initial purification, add lectin to the exosome suspension, and use a magnetic stirrer to stir...

Embodiment 2

[0027] A method for isolating and purifying exosomes, specifically according to the following steps:

[0028] S1: Material preparation, 60ml of biological fluid, 1 differential centrifuge tube, 1 purification column, 1 affinity chromatography column, 35g of hydroxyapatite, 50ml of sodium dihydrogen phosphate solution, 30ml of lectin, 70ml of sucrose solution, Phosphate buffered saline solution, 1 storage tank, 1 magnetic stirrer, 1 sterile filter;

[0029] S2: For the first separation, put the biological fluid into a differential centrifuge tube, and centrifuge at 400g for 4min, 4000g for 4min, and 20000g for 4min to remove cells, dead cells and cell debris respectively; then use ultra-high speed centrifugation to obtain exocrine The crude extract of exosomes was centrifuged at 200,000g for 4min to remove contaminating proteins to obtain exosome suspension; the exosomes were separated by differential centrifugation;

[0030] S3: For the initial purification, add lectin to the...

Embodiment 3

[0036] A method for isolating and purifying exosomes, specifically according to the following steps:

[0037] S1: Material preparation, biological fluid 100ml, 1 differential centrifuge tube, 1 purification column, 1 affinity chromatography column, 50g of hydroxyapatite, 80ml of sodium dihydrogen phosphate solution, 40ml of lectin, 110ml of sucrose solution, Phosphate buffered saline solution, 1 storage tank, 1 magnetic stirrer, 1 sterile filter;

[0038] S2: For the first separation, put the biological fluid into a differential centrifuge tube, and centrifuge at 500g for 5min, 5000g for 5min, and 30000g for 5min to remove cells, dead cells and cell debris respectively; then use ultra-high speed centrifugation to obtain exocrine The crude extract of exosomes was centrifuged at 300,000g for 5min to remove contaminating proteins to obtain exosome suspension; the exosomes were separated by differential centrifugation;

[0039]S3: For the initial purification, add lectin to the e...

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Abstract

The invention discloses a method for separating and purifying exosomes. The method comprises the following steps: S1, preparing materials; S2, performing primary separation; S3, performing primary purification; S4, performing filtering and purifying to obtain exosomes with relatively high purity, repeatedly performing filtering twice by using a sterile filter, and filtering impurities to obtain high-purity exosomes; S5, strengthening purification; S6, performing standing and mixing; and S7, collecting the exosomes. By adding agglutinin, a phosphate buffer salt solution, a sucrose solution and an exosome suspension for a cooperative reaction, the purification degree of the exosomes is gradually increased, the operation is simpler, the purification speed is high, filtration is performed through the sterile filter, mixed impurity proteins in the exosomes are effectively removed, the purity and yield of the exosomes are ensured, and a large quantity of polluted proteins in the exosomes are removed through hydroxyapatite and sodium dihydrogen phosphate solutions, so that the purification quality is further guaranteed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for isolating and purifying exosomes. Background technique [0002] Exosomes are 30-200nm-diameter encapsulated cystic vesicles with double-layered lipid molecules, which are selectively packaged and released by living cells, and are abundant in human body fluids. Exosomes contain different types of Lipids, nucleic acids, and proteins, etc., related studies have also shown that exosomes play a huge role in intercellular communication and physiological and pathological processes; at present, the removal of foreign proteins in exosomes is an urgent solution The problem is that in the existing separation and purification methods, the exosome suspension still contains a large amount of impurity proteins, which are not pure exosomes, and the purification degree is low and the speed is slow. Contents of the invention [0003] The purpose of the present invention is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00
CPCC12N5/00C12N2509/00C12N2509/10
Inventor 丁小梅
Owner 中聚生物技术(广东)集团有限公司
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