Application of noninvasive ultrasonic processing of cells to preparation of exosomes, exosomes and preparation method and application of exosomes

A technology of ultrasonic treatment and exosomes, which is applied to exosomes and their preparation. The application field of non-invasive ultrasonic treatment of cells in the preparation of exosomes can solve the problem of difficulty in realizing the expression regulation of multiple target substances, high cost, and complicated operation. problems, to achieve the effect of shortening preparation time, improving expression, and increasing expression abundance

Active Publication Date: 2019-04-02
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first purpose of the present invention is to provide a preparation method of exosomes and exosomes, so as to alleviate the technical problems of high cost, complicated operation and difficulty in realizing the expression regulation of multiple target substances in the preparation method of exosomes in the prior art

Method used

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  • Application of noninvasive ultrasonic processing of cells to preparation of exosomes, exosomes and preparation method and application of exosomes
  • Application of noninvasive ultrasonic processing of cells to preparation of exosomes, exosomes and preparation method and application of exosomes
  • Application of noninvasive ultrasonic processing of cells to preparation of exosomes, exosomes and preparation method and application of exosomes

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Experimental program
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preparation example Construction

[0050]A method for preparing exosomes, comprising the following steps: treating cells with non-invasive ultrasound, and extracting exosomes.

[0051] The exosome preparation method provided by the present invention uses non-invasive ultrasonic treatment of cells, and regulates the expression abundance of microRNA in cell exosomes, and / or potentially increases the expression of brain-derived neurotrophic factor by stimulating the cells to a certain extent , and / or promote the removal of harmful proteins from the brain, and / or reduce the oxidative damage of harmful substances to brain tissue. This method is applicable to all cells, such as tumor cells, stem cells, nerve cells, and osteoarthritis cells. The target exosomes can be obtained within a short period of time, which greatly shortens the preparation time of exosomes. In addition, this method can simultaneously increase the expression abundance of multiple microRNAs. Compared with the gene transfection method, more exosom...

Embodiment 1

[0074] Example 1 Culture of cells in serum-free medium and non-invasive stimulation of cells by ultrasound

[0075] In this embodiment, SH-SY5Y cells are used. After the SH-SY5Y cells are cultured to a confluence of 70%-80%, they are replaced with serum-free high-glucose DMEM medium.

[0076] The ultrasonic device stimulates the cell culture dish, and the total ultrasonic stimulation time lasts for 5 minutes. The specific ultrasonic stimulation method is as follows: figure 1 shown. Specific ultrasonic parameters, probe frequency: 1MHz, amplitude: 100mV, ultrasonic time: 5 minutes.

[0077] After the stimulation, the morphology of the cells was observed under a microscope, and the results showed that the cells after ultrasonic treatment were in good shape, which was no different from that of unstimulated SH-SY5Y cells.

[0078] The sonicated SH-SY5Y cells were continued to be cultured in a 37° incubator, and after 24 hours and 48 hours of culture, the cell culture supernatant...

Embodiment 2

[0079] Example 2 Exosome collection after ultrasonic stimulation

[0080] The flow chart of exosome extraction is as follows: figure 2 As shown, specifically:

[0081] 1) Centrifuge the cell culture supernatant collected in Example 1 at 300×g for 5 minutes to remove the cells;

[0082] 2) Centrifuge the solution supernatant in step 1) at 2000×g for 10 minutes to remove apoptotic bodies;

[0083] 3) Concentrate the solution in step 2) with a 3kDa ultrafiltration tube, centrifuge at 3000×g for 30 minutes, and collect the retentate;

[0084] 4) Mix the concentrated medium in step 3) with 16% (w / v) polyethylene glycol at a volume ratio of 1:1, and place the mixed liquid at 4°C for 24 hours to precipitate to obtain the precipitate;

[0085] 5) The precipitate in step 4) was centrifuged at 100,000×g for 2 hours, and the precipitate was exosomes.

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Abstract

The invention relates to the technical field of biology, and particularly provides an application of noninvasive ultrasonic processing of cells to preparation of exosomes, the exosomes and a preparation method and application of the exosomes. The preparation method of the exosomes provided by the invention adopts noninvasive ultrasonic processing for the cells, the cells are certainly stimulated,the expression richness of microRNA in the cell exosomes is adjusted and controlled, and/or the expression of brain-derived neurotrophy factors is potentially increased, and/or the elimination of protein from the brain is promoted, and/or the oxidation damage of hazardous substances to brain tissue is alleviated. The method is applicable to all of the cells, is good in universality and simple to operate. The exosomes are high in yield, and can obtain target exosomes within a short time. In addition, through the method, simultaneous increment of the expression richness of microRNA in the exosomes can be realized, the exosomes being high in microRNA expression changes but not the exosomes being changed in single microRNA can be obtained.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, to the application of non-invasive ultrasonic treatment of cells in the preparation of exosomes, exosomes and their preparation methods and applications. Background technique [0002] Exosomes are a multivesicular body that exists outside the cell, and can form multivesicular endosomes through the inward depression of the endocytic vesicle membrane, and release the small vesicles in the endosome after fusion with the cell membrane. Exosomes have a diameter of 30-150nm, contain RNA, protein, microRNA, DNA fragments and other components, and are distributed in various body fluids such as blood, saliva, urine, cerebrospinal fluid and breast milk. microRNA is a kind of endogenous non-coding RNA with regulatory function, which can bind to the 3'untranslated region of the target mRNA, resulting in differential expression of the target gene. Therefore, exosomes can deliver microRNA to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N13/00A61K35/30A61P25/28A61P25/16A61P9/10A61P25/00
CPCA61K35/30A61P9/10A61P25/00A61P25/16A61P25/28C12N5/0618C12N13/00
Inventor 郑海荣严飞邓志婷肖杨
Owner SHENZHEN INST OF ADVANCED TECH
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