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Separation and enrichment method for quickly extracting tissue extracellular vesicles

A technology for separation, enrichment, and tissue cells, which is applied in the biological field, can solve the problems of cumbersome operation, long extraction time of tissue extracellular vesicles, and low purity, and achieve the effects of simplified steps, low pollution of soluble impurities, and high purity

Inactive Publication Date: 2021-03-16
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

[0005] The present invention aims at the technical defects and deficiencies of existing tissue extracellular vesicles, such as long extraction time, low purity, cumbersome operation, etc., and provides a rapid extraction based on enzymatic hydrolysis + differential centrifugation + ultracentrifugation + molecular exclusion + ultrafiltration Isolation and enrichment method of tissue extracellular vesicles

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  • Separation and enrichment method for quickly extracting tissue extracellular vesicles
  • Separation and enrichment method for quickly extracting tissue extracellular vesicles
  • Separation and enrichment method for quickly extracting tissue extracellular vesicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Enzymolysis process optimization experiment

[0041] In the early stage of the experiment, papain, collagenase + DNase I, and tissue-digesting enzyme were used to dissociate mouse heart tissue, and the optimal tissue-digesting enzyme was selected by comparing the experimental results, as follows:

[0042] 1. Papain dissolving scheme: the dosage of papain (Worthington-biochemical, LK003178) is 15-30U, and the papain solution used is to dissolve papain with Earle's balanced salt solution (EBSS buffer), and then add Hibernate E medium to obtain wherein, the volume ratio of Earle's balanced salt solution to Hibernate E medium is 1:3, and the amount of papain solution is about 3mL; the extracellular vesicle size analysis diagram of papain is shown in figure 1 As shown in a, the WB of Calnexin protein is shown as figure 2 As shown in a, the mass of mouse heart tissue and the total amount of BCA protein are shown in Table 1.

[0043]2. Collagenase D and DNase I...

Embodiment 2

[0048] Example 2 Enrichment method for rapid extraction of extracellular vesicles from mouse heart tissue

[0049] The method for enriching extracellular vesicles in mouse heart tissue, the specific steps are as follows:

[0050] 1. Cut the cryogenically frozen (-80°C) tissue into 1-2 mm with a blade 3 size;

[0051] 2. The tissue slices were transferred to a centrifuge tube containing 2.5 mL of tissue-digesting enzyme dissociation solution, wherein the preparation of the tissue-digesting enzyme was the same as in Example 1;

[0052] 3. Put the above mixture into a 37°C water bath, mix it every 5 minutes, and dissociate for 30 minutes to obtain a tissue dissociation solution, and then filter the above tissue dissociation solution with a 70 μm filter membrane into a new centrifuge tube to obtain a tissue cell suspension;

[0053] 4. Add 25uL protease and phosphatase inhibitor mixture to the tissue cell suspension;

[0054] 5. Centrifuge the mixture in step 4 at 300-600 g fo...

Embodiment 3

[0063] Example 3 Enrichment method for rapid extraction of extracellular vesicles from mouse liver tissue

[0064] The method for enriching extracellular vesicles in mouse liver tissue, the specific steps are as follows:

[0065] 1. Cut the cryogenically frozen (-80°C) tissue into 1-2 mm with a blade 3 size;

[0066] 2. The tissue slices were transferred to a centrifuge tube containing 3mL of tissue-digesting enzyme dissociation solution, wherein the preparation of tissue-digesting enzyme was the same as in Example 1;

[0067] 3. Put the above mixture into a 37°C water bath, mix it every 5 minutes, and dissociate for 20 minutes to obtain a tissue dissociation solution, and then filter the above tissue dissociation solution into a new centrifuge tube with a 70 μm filter membrane to obtain a tissue cell suspension;

[0068] 4. Add 30uL protease and phosphatase inhibitor mixture to the tissue cell suspension;

[0069] 5. Centrifuge the mixture in step 4 at 300-600g for 10min ...

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Abstract

The invention provides a separation and enrichment method for quickly extracting tissue extracellular vesicles. The separation and enrichment method comprises the following steps of: A: shredding target tissues by a mechanical method, adding tissue digestive enzymes to carry out tissue dissociation, and filtering obtained tissue dissociation liquid to obtain a tissue cell suspension; and B: carrying out differential centrifugation, ultracentrifugation, SEC exclusion and ultrafiltration on the tissue cell suspension in sequence, and carrying out enrichment and purification of the tissue extracellular vesicles. The tissue extracellular vesicle separation and enrichment method provided by the invention greatly simplifies a step of separating the tissue extracellular vesicles, enrichment timeis saved, and a whole process only needs 4-5h. The enriched extracellular vesicle has high purity, small soluble impure protein pollution and wide practicability. The extracellular vesicle enriched bya tiny quantity of tissue samples can meet subsequent analysis, including NTA, WB, electron microscopes, transcriptomes and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a separation and enrichment method for rapidly extracting tissue extracellular vesicles. Background technique [0002] Extracellular vesicles are lipid bilayer membrane-coated particles with a size of 30-150 nm released by different cells. They can wrap and deliver molecules such as RNA and protein, and participate in various physiological and pathological pathways as messengers of intercellular communication. At present, extracellular vesicles have been successfully isolated from body fluids such as blood, urine, and saliva, but there are few related studies on the isolation of extracellular vesicles from tissue intercellular spaces. The key steps in the extraction and separation of extracellular vesicles in the interstitial space are shearing and enzymatic hydrolysis to obtain a cell suspension. This process will cause a certain degree of damage to the cell structure. The ruptured...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2509/00C12N2509/10
Inventor 王程程秦伟伟程敏林海军孔关义
Owner 北京恩泽康泰生物科技有限公司
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