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Method and kit for separating extracellular vesicles of tissue specific origin

A technology of cells and vesicles, applied in the field of separating tissue-specific extracellular vesicles, which can solve the problems of low recovery, low purity, and poor dispersion, and achieve stable recovery, simple operation, and good repeatability

Active Publication Date: 2020-04-28
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have brought a series of unique advantages to exosomes, but there are still major deficiencies, such as low purity, low recovery, poor dispersion, damage to integrity, and difficulty in widespread popularization. The most important thing is that they cannot be effectively enriched. Exosomes Accumulated to Tissue or Disease Origin

Method used

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  • Method and kit for separating extracellular vesicles of tissue specific origin
  • Method and kit for separating extracellular vesicles of tissue specific origin
  • Method and kit for separating extracellular vesicles of tissue specific origin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Specific extracellular vesicle / exosome capture and isolation method

[0104] 1.1 Sample pretreatment and obtain total extracellular vesicles or total exosomes without background interference

[0105] 1.1.1 Enrichment of total extracellular vesicles or total exosomes

[0106] Total extracellular vesicles or total exosomes were extracted and purified from samples (blood, urine, pleural effusion, cell supernatant, pleural effusion, alveolar lavage fluid) by PEG precipitation or molecular chromatography exclusion (SEC). Specific steps are as follows:

[0107] 1) Enrichment of total extracellular vesicles by PEG precipitation

[0108] Add 120 μL PEG8000 precipitant to 500 μL serum / plasma, add 60 μL PEG8000 precipitant to 250 μL pleural and peritoneal fluid; mix upside down 5 times, and let stand at 4-8°C for 30 minutes; then centrifuge at 13000 rpm for 2 minutes, discard the supernatant to retain the precipitate, Add 100 μL PBS to resuspend and mix well to obtai...

Embodiment 2

[0143]Example 2 Comparison of Specific Exosome Capture and Separation Method System Optimization

[0144] 2.1 Capture carrier optimization

[0145] 2.1.1 Capture carrier magnetic bead screening

[0146] Considering the principles of magnetic bead dispersion, sedimentation, non-specific adsorption, etc., and considering that the larger the particle size of the magnetic bead (the larger the specific surface area, the more sites that bind to the target), the capture efficiency will be higher. Therefore, two magnetic beads with a particle size of more than 1 μm and mature commercialization were selected for comparison and verification.

[0147] The verification method and steps are as follows:

[0148] 1) Take 10 μL of each of the two types of magnetic beads and incubate with 30 μg of exosomes at room temperature (10 rpm) for 15 minutes, then dilute to 1 mL with PBS and rotate at room temperature for 30 minutes

[0149] 2) Termination reaction: PBS containing 100mM glycine, 2%B...

Embodiment 3

[0227] Example 3: Validation of the capture system effect

[0228] 3.1. Thyroid transcription factor (TTF-1) captures exosomes from lung tissue and thyroid tissue

[0229] The verification method is as follows:

[0230] 1) Lung cancer, intestinal cancer, breast cancer, thyroid cancer, liver cancer and gastric cancer cells (purchased by the Chinese Academy of Sciences Cell Bank) were starved for culture, and supernatant total exosomes were enriched by ultracentrifugation

[0231] 2) TTF-1-positive exosomes from exosome samples secreted by different tumor cell supernatants were captured by the resin method (the method is the same as part 1.3 of Example 1)

[0232] Western Blot (WB) was used to detect the target protein TTF-1 exosome protein to verify whether TTF-1 positive exosomes could be captured in samples from different tissue sources.

[0233] Such as Figure 7 As shown, WB detection after TTF-1 affinity capture of exosomes in tumor cell supernatant, TTF-1 positive exos...

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Abstract

The present application provides a method and kit for separating extracellular vesicles of tissue specific origin, and specifically, provides the method for separating the thyroid transcription factor1 positive extracellular vesicles, especially exosomes, from a biological fluid. The method includes the following steps: optionally separating total extracellular vesicles by a physical process, optionally removing extracellular vesicles derived from red blood cells and / or white blood cells by a binding agent, and positively capturing the extracellular vesicles or exosomes of target cells by using a binding agent binding to the thyroid transcription factor 1. The present application also provides an application of the binding agent binding to thyroid transcription factor 1 in preparation ofseparation or detection reagents of the extracellular vesicles or the exosomes, and the kit for separating the thyroid transcription factor positive extracellular vesicles or exosomes from the biological fluid.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, the present application relates to methods for isolating extracellular vesicles, such as exosomes, of tissue-specific origin. Background technique [0002] Extracellular vesicles (Extracellular Vesicles, EVs) are also called extracellular vesicles, including microvesicles, exosomes, etc. The secreted form is a vesicle with a size of about 30-100 nm released outside the cell, and a microvesicle is a vesicle with a size of about 100-1000 nm. Extracellular vesicles have a lipid bilayer membrane structure and are widely found in all body fluids, including saliva, blood, urine, and breast milk. Current studies have found that exosomes are rich in nucleic acids (microRNA, lncRNA, circRNA, mRNA, tRNA, etc.), protein, cholesterol, etc., which can be transmitted to target cells as signal molecules, thereby mediating material transfer and information exchange between cells, regulat...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/6886C12N15/11
CPCC12N5/0688C12N5/0617C12Q1/6886C12N2509/00C12Q2600/158C12Q2600/178
Inventor 渠香云董肇楠贾云莉马雪情李若晨蒋晓旭王弢
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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