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Method for extracting extracellular vesicles of soft tissue and application

An extraction method and soft tissue technology, applied in the field of soft tissue extracellular vesicle extraction, can solve the problems of less extraction and inaccurate results, and achieve the effects of less impurity protein, simple extraction and cost saving.

Active Publication Date: 2020-09-04
成都世联康健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the above technical problems, the present invention provides a method for extracting extracellular vesicles from soft tissues. In view of the shortcomings in the extraction of exosomes or microvesicles derived from the above-mentioned cell culture supernatant, the amount extracted during the diagnosis process is small and the results are low. Inaccurate technical problems, provide a method for extracting exosomes or microvesicles directly from the soft tissue culture supernatant; the present invention carries out tissue culture of soft tissues (such as placenta, umbilical cord, fat, muscle, etc.) in a healthy state or pathological changes , to extract exosomes or microvesicles, used in disease treatment, disease diagnosis, medical care, etc.

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  • Method for extracting extracellular vesicles of soft tissue and application
  • Method for extracting extracellular vesicles of soft tissue and application
  • Method for extracting extracellular vesicles of soft tissue and application

Examples

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Effect test

Embodiment 1

[0034] A method for extracting soft tissue extracellular vesicles, the specific steps are as follows:

[0035] (1) Obtain the target soft tissue, wash it and cut it into small pieces;

[0036] (2) Transfer the shredded tissue pieces to a suspension culture bottle, add α-MEM (hyclone) basal medium, the mass volume ratio of the tissue pieces to the basal medium is 1:15, and culture them in an incubator, magnetic The speed of the stirrer is 100rpm, the cultivation temperature is 37°C, and the cultivation time is 40h;

[0037] (3) Transfer the α-MEM tissue culture supernatant to a centrifuge tube or an EP tube, centrifuge at 1000g for 10min at 4°C;

[0038] (4) Transfer the tissue culture supernatant after centrifugation to an ultrafiltration tube, centrifuge at 3000g for 30min at 4°C to obtain a retentate;

[0039] (5) add retentate to chromatography in the chromatography column, collect components;

[0040] (6) Collect the retentate after chromatography in step (5), store it ...

Embodiment 2

[0042] A method for extracting soft tissue extracellular vesicles, the specific steps are as follows:

[0043](1) Obtain the target soft tissue, put the tissue into tissue preservation solution containing 1% (vol / vol) penicillin / streptomycin; -Mixed in MEM basal medium; store the tissue at -196°C; wash and cut into small pieces when used;

[0044] (2) Transfer the shredded tissue pieces to a suspension culture bottle, add α-MEM basal medium, the mass volume ratio of tissue pieces to basal medium is 1:25, and culture them in an incubator with a magnetic stirrer at The temperature is 200rpm, the culture temperature is 36°C, and the culture time is 60h;

[0045] (3) Transfer the α-MEM tissue culture supernatant to a centrifuge tube or an EP tube, centrifuge at 2000g for 8min at a temperature of 5°C;

[0046] (4) Transfer the tissue culture supernatant after centrifugation to an ultrafiltration tube, centrifuge at 5000g for 40min at a temperature of 5°C, and obtain a retentate; ...

Embodiment 3

[0050] A method for extracting soft tissue extracellular vesicles, the specific steps are as follows:

[0051] (1) Obtain the target soft tissue, put the tissue into the tissue preservation solution containing 1% (vol / vol) penicillin / streptomycin; the tissue preservation solution is 1mL penicillin / streptomycin solution (hyclone) mixed with 100mL α-MEM Prepared; store the tissue at -20°C and send it to the processing laboratory as soon as possible (within 72 hours); wash it and cut it into small pieces;

[0052] (2) Transfer the shredded tissue pieces to a suspension culture bottle, add α-MEM basal medium, the mass volume ratio of tissue pieces to basal medium is 1:10, and culture them in an incubator with a magnetic stirrer at The temperature is 300rpm, the culture temperature is 38°C, and the culture time is 70h;

[0053] (3) Transfer the α-MEM tissue culture supernatant to a centrifuge tube or an EP tube, centrifuge at 1500g for 25min at 4°C;

[0054] (4) After centrifugat...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a method for extracting extracellular vesicles of soft tissue and an application. The method comprises the steps ofobtaining an appropriate amount of target soft tissue from an organism, performing storage, performing culture, performing chromatography and the like. According to the method disclosed by the invention, extraction of exosomes or microvesicles of the soft tissue is simple, cell separation and purification processes are avoided, time and labor are saved and the cost is reduced. Furthermore, the physiological characteristics of in vivo soft tissue can be really reflected, the extracellular vesicles can be applied to disease diagnosis as effective physiological detection markers, and the defect in the exosomes or microvesicles of a body fluid or cell culture supernatant source is compensated.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for extracting soft tissue extracellular vesicles and its application. Background technique [0002] Extracellular Vesicles (EVs), referred to as EVs, is a collective term for various vesicle structures with membrane structures released by cells. The diameter of these vesicles can range from 30, 40nm to 8, 9um. The vesicles with a double-membrane structure that are shed from the cell membrane or secreted by the cell carry a variety of proteins, lipids, DNA, mRNA, miRNA and other non-coding RNAs (non-coding RNAs, ncRNAs) from the donor cell ). Extracellular vesicles are tools for information transmission between cells (cell communication), which regulate cell proliferation, differentiation, immune regulation, tumor killing, cell migration, angiogenesis and other processes; in addition, extracellular vesicles secreted by tissue cells into the blood Vesicles can also serve as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/073A61K8/99A61K35/34A61K35/51A61K35/50A61K47/46A61Q19/00
CPCC12N5/0658C12N5/0605C12N5/0603A61K8/99A61K35/34A61K35/51A61K35/50A61K47/46A61Q19/00C12N2509/00C12N2509/10
Inventor 乔鞠廖立黄海森陈国庆
Owner 成都世联康健生物科技有限公司
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