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Isolation method of stem cell exosomes

A separation method and stem cell technology, applied in the field of separation of stem cell exosomes, can solve the problems of high exosome concentration and expensive equipment, and achieve the effect of retaining activity, short centrifugation time and simple separation method

Active Publication Date: 2019-09-17
金银鹏 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Exosome separation methods in the prior art include: (1) ultra-high-speed centrifugation or density gradient centrifugation. This method has a limiting factor, that is, it needs to be equipped with various ultra-high-speed high-speed machines, and the price of this equipment is Very expensive, thus limiting the research on exosomes in some small and medium-sized laboratories; (2) using ultrafiltration method, the concentration of exosomes collected by simple ultrafiltration method cannot reach very high, for exosomes requiring higher concentration Applications such as body detection and treatment are also a limitation

Method used

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  • Isolation method of stem cell exosomes
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  • Isolation method of stem cell exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Isolation and culture of hASCs stem cells

[0029] Experimental method: Take adipose tissue after liposuction from a young healthy human body, rinse it with PBS solution 3 times and cut it into about 1mm 3 Add 0.1% collagenase I to digest the small pieces for 30 minutes. Prepare complete medium: 500ml medium, 10% serum, 10ng / ml bFGF, add an equal volume of complete medium to stop the digestion, centrifuge at 1000rpm for 10min. Resuspend cells, filter with 100μm filter and press 1×10 6 / ml inoculated in T25 cell culture flask, set 37℃, 5% CO 2 Incubate in a saturated humidity incubator and change the medium after 48 hours. Observe that the cells are 90% confluent, digested with 0.25% trypsin, and subcultured at 1:3.

[0030] Flow cytometry detects the surface molecular markers of hASCs: CD44, CD73, CD90, CD105.

[0031] Experimental results: After the P2 generation, the isolated hASCs maintained a fibroblast-like morphology, uniform in size and densely arranged, gr...

Embodiment 2

[0035] This embodiment provides a method for isolating stem cell exosomes, which includes the following steps:

[0036] S1: Use serum-containing medium to cultivate stem cells to a cell density of 80%, and collect stem cells; use washing solution to wash stem cells 3 times, then use serum-free medium to continue culturing stem cells for 24 hours; among them, serum-containing medium has a mass fraction of 10 % FBS in DMEM medium; Serum-free medium is DMEM medium; washing solution includes: 0.01M PBS buffer, the pH of PBS buffer is 7.2;

[0037] S2: Remove the stem cells in the culture system obtained by S1, then filter the remaining components with a 0.22μm filter membrane, and collect the filtrate;

[0038] S3: Add the filtrate to a 15mL 10KD concentration tube (the first concentration tube), and centrifuge at 4000g for 40 minutes at 4°C to obtain a concentrated solution;

[0039] S4: Dispense the concentrated solution into a 0.5mL 10KD concentration tube (second concentration tube), ...

Embodiment 3

[0042] This embodiment provides a method for isolating stem cell exosomes, which includes the following steps:

[0043] S1: Use serum-containing medium to cultivate stem cells to a cell density of 70%, and collect stem cells; wash stem cells twice with washing solution, and then use serum-free medium to continue culturing stem cells for 23 hours; among them, serum-containing medium contains a mass fraction of 10% FBS in DMEM medium; serum-free medium is DMEM medium; washing solution includes: 0.01M PBS buffer, the pH of PBS buffer is 7.2;

[0044] S2: Remove the stem cells in the culture system obtained by S1, then filter the remaining components with a 0.22μm filter membrane, and collect the filtrate;

[0045] S3: Add the filtrate to a 15mL 10KD concentration tube (the first concentration tube), and centrifuge at 3500g for 35 minutes at 4°C to obtain a concentrated solution;

[0046] S4: Dispense the concentrated solution into 0.5mL 10KD concentration tube (second concentration tube)...

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Abstract

The invention relates to a separation method of stem cell exosomes. The method includes: using a culture medium containing serum to culture stem cells until cell density is larger than 70%, collecting the stem cells, and then using a culture medium which does not contain the serum to culture the stem cells; collecting the culture medium in obtained products, filtering, and collecting filtrate; adding the filtrate into a first concentrating tube, and centrifuging to obtain a concentrated solution; split charging the concentrated solution into a second concentrating tube, and centrifuging; adding a buffer solution into the second concentrating tube, and centrifuging again; inversely placing the inner tube of the second concentrating tube after the second centrifuging into a collecting tube, and centrifuging, wherein liquid in the collecting tube is a solution containing high-concentration stem cell exosomes. The method has the advantages that the concentrating tubes of different sizes are used in a combined manner to fast, efficiently and cheaply extract the exosomes in the culture medium, and the method is short in centrifuging time, capable of effectively reducing the mechanical damage of the exosomes and capable of well keeping the activity of the exosomes.

Description

Technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for separating stem cell exosomes. Background technique [0002] Exosomes are membranous vesicles with a diameter of about 30-150nm that are released into the extracellular matrix after the fusion of multivesicular bodies (MVB) in the cell and the cell membrane. Many cells can secrete exosomes, such as T cells, B cells, dendritic cells, mast cells and tumor cells. Exosomes contain a variety of proteins and RNAs, which play a role in communicating information and mutual regulation between cells. However, the method of transmitting information and the mechanism of mutual regulation are not completely clear, especially in tumor cells. Between: whether the exosomes transmit the characteristic information of cancer cells to the adjacent tissues, and promote their canceration through the proteins they contain; whether the exosomes have an influence on the migration, prolifera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 金银鹏傅青春李洪超王俊怡王皙张苗孟玲玉王晓今陈成伟
Owner 金银鹏
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