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Extraction process of extracellular exosomes based on differential centrifugation

A differential centrifugation and extraction technology, applied in artificial cell constructs, animal cells, cell dissociation methods, etc., can solve the problem of low purity of exosomes, achieve good extraction effect, promote development and growth, and improve purity. Effect

Inactive Publication Date: 2020-01-10
赵凯
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies of the prior art, the present invention provides a cell exosome extraction process based on differential centrifugation, which solves the problem of low purity of the exosomes extracted by the prior art cell exosome extraction method

Method used

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Examples

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Comparison scheme
Effect test

Embodiment 1

[0027] A process for extracting exosomes based on differential centrifugation in this embodiment comprises the following steps:

[0028] S1. Culture HEK-293 cells in DMEM medium, add 10% horse serum to the medium before use, then place the cells in an incubator containing 5% CO2, and pass passage once every 4 days;

[0029] S2. Observe the growth of HEK-293 cells in S1. Discard the original medium when the cell confluence reaches about 90%, wash with PBS buffer three times, add DMEM medium, and place it at 37°C Under culture for 36h, collect the supernatant after culture;

[0030] S3. Take the supernatant in 100mL of S2 and centrifuge at 3000×g for 18 minutes at 4°C to remove dead cells, transfer the remaining supernatant to a centrifugal ultrafilter, and concentrate at 4°C with a horizontal rotor at 4000×g for about to 1 mL;

[0031] S4. Centrifuge the supernatant after S3 treatment at 10,000×g for 40 minutes to remove impurities such as cell debris and dead cells, such as ...

Embodiment 2

[0042] A process for extracting exosomes based on differential centrifugation in this embodiment comprises the following steps:

[0043] S1. HEK-293 cells were cultured in DMEM medium, and 10% horse serum was added to the medium before use, and then the cells were cultured in an incubator containing 5% CO2, and passaged once every 5 days;

[0044] S2. Observe the growth of HEK-293 cells in S1. Discard the original medium when the cell confluence reaches about 90%, wash with PBS buffer three times, add DMEM medium, and place it at 37°C Under culture for 24 hours, after the culture, the supernatant was collected;

[0045] S3. Take the supernatant in 100mL of S2 and centrifuge at 3000×g at 4°C for 15 minutes to remove dead cells, transfer the remaining supernatant to a centrifugal ultrafilter, and concentrate at 4°C with a horizontal rotor at 4000×g for about to 1 mL;

[0046] S4. Centrifuge the supernatant after S3 treatment at 10,000×g for 30 minutes to remove impurities such...

Embodiment 3

[0057] A process for extracting exosomes based on differential centrifugation in this embodiment comprises the following steps:

[0058] S1. HEK-293 cells were cultured in DMEM medium, and 10% horse serum was added to the medium before use, and then the cells were cultured in an incubator containing 5% CO2, and passaged once every 6 days;

[0059] S2. Observe the growth of HEK-293 cells in S1. Discard the original medium when the cell confluence reaches about 90%, wash with PBS buffer three times, add DMEM medium, and place it at 37°C Cultivate for 30 h, and collect the supernatant after culturing;

[0060] S3. Take the supernatant in 100mL of S2 and centrifuge at 3000×g for 20min at 4°C to remove dead cells, transfer the remaining supernatant into a centrifugal ultrafilter, and centrifuge at 4000×g in a horizontal rotor at 4°C to concentrate for about to 1 mL;

[0061] S4. Centrifuge the supernatant after S3 treatment at 10,000×g for 35 minutes to remove impurities such as ...

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Abstract

The invention relates to the technical field of exosome extraction technology, and provides an extraction process of extracellular exosomes based on differential centrifugation. The culture for culturing HEK-293 cells is preferably a high-glucose DMEM culture medium, the problem that the adhesion strength of the HEK-293 cells is relatively small during the culture process can be effectively solved, so that the HEK-293 cells in the culture medium are produced at the same location, and the HEK-293 cells are isolated conveniently; in the culture process of the HEK-293 cells, 10% of horse serum isadded to the DMEM culture medium, a similar biological internal environment can be provided for cells cultured in vitro, the horse serum further contains several animal hormones and enzymes, and thedevelopment and growth of the HEK-293 cells can be promoted; and step S1, step S2 and step S3 are carried out on the HEK-293 cells before the differential centrifugation is performed, a supernatant extracted from the cells can be concentrated, the supernatant is collected after concentration, the purity of the exosomes in an extract can be improved to a certain extent, and the extraction effect isbetter.

Description

technical field [0001] The invention relates to the technical field of exosome extraction technology, in particular to a cell exosome extraction process based on differential centrifugation. Background technique [0002] Exosomes, Chinese name exosome, exosome, secretory body, are a kind of tiny membrane vesicles that can be secreted by almost all living cells, and have a lipid bilayer membrane structure. Exosomes contain a large number of proteins and non-coding RNAs such as mRNA, miRNA, IncRNA, and dircRNA, which carry and transmit important signaling molecules, forming a new cell-to-cell information transmission system that affects the physiological state of cells and communicates with them. The occurrence and process of many diseases are closely related. Its function depends on the cell type it is derived from, and it can participate in the body's immune response, antigen presentation, cell migration, cell differentiation, tumor invasion and other aspects. There are CD...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2509/00
Inventor 赵凯
Owner 赵凯
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