Production method of exosome carrying plasmid

A production method and technology for exosomes, applied in the field of exosomes, can solve problems such as damage to the surface properties of exosomes, and the efficiency of injection needs to be improved.

Active Publication Date: 2021-12-17
THE UNIV OF HONG KONG SHENZHEN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, using electroporation, miRNA can be injected into exosomes, but it may damage the original surface properties of exosomes, and the efficiency of injection needs to be improved.

Method used

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  • Production method of exosome carrying plasmid
  • Production method of exosome carrying plasmid
  • Production method of exosome carrying plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 4

[0041] A method for producing exosomes carrying a plasmid provided in Example 4 of the present invention is implemented through the following steps:

[0042] S1. Transfection method: culture human-derived bone marrow mesenchymal stem cells in vitro in a T75 culture flask until the fusion reaches 85%, discard the culture supernatant, add 6ml Opti-MEM, place in cell culture for cultivation, and obtain cells containing Opti-MEM Human-derived mesenchymal stem cells in culture medium. The plasmid-liposome complex obtained in Example 1 of the present invention was added to human-derived mesenchymal stem cells containing Opti-MEM cell culture medium, shaken crosswise, and placed in a cell incubator for transfection culture for 6 hours;

[0043] S2, lysosome inhibition: after the transfection is completed, remove the supernatant with the plasmid-liposome complex, add α-MEM medium containing 10% exosome-free fetal bovine serum and chloroquine at a concentration of 25uM Or chloroquine ...

Embodiment 5

[0046] A method for producing exosomes carrying a plasmid provided in Example 5 of the present invention is implemented through the following steps:

[0047]S1. Transfection method: culture human-derived bone marrow mesenchymal stem cells in vitro in a T75 culture flask until the fusion reaches 95%, discard the culture supernatant, add 6ml Opti-MEM, place in cell culture for cultivation, and obtain cells containing Opti-MEM Human-derived mesenchymal stem cells in culture medium. The plasmid-liposome complex obtained in Example 1 of the present invention was added for cultivation to obtain human-derived mesenchymal stem cells containing Opti-MEM cell culture medium, shaken crosswise, and placed in a cell culture incubator for transfection and culture for 6 hours ;

[0048] S2, lysosome inhibition: after the transfection is completed, remove the supernatant with the plasmid-liposome complex, add α-MEM medium containing 10% exosome-free fetal bovine serum and chloroquine at a co...

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Abstract

The invention discloses a production method of an exosome carrying plasmid. The method comprises the following steps of: 1) fusing human mesenchymal stem cells cultured in vitro to 85-95%; 2) discarding the culture supernatant, adding a plasmid liposome compound, and performing transfection culture in a cell incubator; 3) after the transfection culture is completed, removing the supernatant carrying the plasmid liposome compound, adding an alpha-MEM culture medium containing exosome-free fetal calf serum and chloroquine or chloroquine phosphate solution, and culturing in the cell incubator; and 4) collecting the culture supernatant, and separating and purifying by using ultracentrifugation or an exosome extraction kit to obtain the exosome carrying plasmid. The exosome obtained by the production method disclosed by the invention can be used as a novel drug, and can be widely used for treating various diseases by carrying different plasmids.

Description

technical field [0001] The invention belongs to the technical field of exosomes, and in particular relates to a production method of exosomes loaded with plasmids. Background technique [0002] Gene therapy is a treatment method that delivers and expresses exogenous genes into dysfunctional cells to repair cell functions and ultimately cure diseases. The effect of gene therapy depends not only on the delivered gene sequence, but also on the efficiency and safety of the carrier. Traditional vectors include viral vectors and non-viral vectors. Viral vectors may undergo insertional mutation, and it is difficult for non-viral vectors to achieve low toxicity and high transfection efficiency at the same time. Therefore, there is a need to develop novel transgenic vectors. [0003] Exosomes are outer membrane vesicles with a diameter of 30-150 nm, which play an important role in the exchange of information between cells. Exosomes are similar to liposomes in non-viral vectors, b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A61K47/46
CPCC12N15/85C12N5/0662A61K47/46C12N2800/107C12N2510/00C12N2501/999Y02A50/30
Inventor 黄德民吴隽李黛叶赵晓丽郝浏智
Owner THE UNIV OF HONG KONG SHENZHEN HOSPITAL
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