CN113801894A - Production method of plasmid-carried exosome - Google Patents
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Abstract
The invention discloses a method for producing plasmid-carried exosomes, which comprises the following steps: 1) culturing in vitro human mesenchymal stem cells to fuse to 85-95%; 2) discarding the culture supernatant, adding the plasmid liposome complex, and placing in a cell culture box for transfection culture; 3) after the transfection culture is finished, removing the supernatant fluid with the plasmid liposome compound, adding an alpha-MEM culture medium containing the fetal calf serum without the exosome and chloroquine or chloroquine phosphate solution, and placing the mixture in a cell culture box for culture; 4) collecting culture supernatant, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the exosome carrying the plasmid. The exosome obtained by the production method can be used as a novel medicine, and can be widely used for treating various diseases by carrying different plasmids.
Description
Technical Field
The invention belongs to the technical field of exosomes, and particularly relates to a production method of an exosome carrying plasmid.
Background
Gene therapy is a therapeutic method in which an exogenous gene is delivered into a dysfunctional cell and expressed to repair the cell function, and finally, the disease is cured. The effectiveness of gene therapy depends on both the gene sequence delivered and the efficiency and safety of the vector. Conventional vectors include viral vectors and non-viral vectors. Viral vectors may have insertional variations, and non-viral vectors have difficulty achieving both low toxicity and high transfection efficiency. Therefore, development of novel transgenic vectors is required.
Exosomes are outer-membrane vesicles 30-150nm in diameter, and play an important role in intercellular information communication. Exosomes are similar to liposomes in non-viral vectors, but are more complex in structure and function and are therefore considered as potential transgene vectors. However, how to use exosomes to package gene sequences efficiently and nondestructively is still an unsolved technical problem. If the miRNA is injected into the exosome by electroporation, the original surface properties of the exosome may be damaged, and the injection efficiency needs to be improved.
Disclosure of Invention
In view of the above, the present invention provides a method for producing plasmid-loaded exosomes, which utilizes mesenchymal stem cells to produce plasmid encapsulated by exosomes, and utilizes chloroquine or chloroquine phosphate to inhibit lysosomes, thereby improving the production efficiency of plasmid encapsulated by exosomes. These exosomes are non-toxic and can be efficiently taken up by recipient cells, allowing the plasmids to be expressed in the recipient cells and exerting a therapeutic effect.
In order to solve the problems, the technical scheme adopted by the invention is a method for producing plasmid-carrying exosomes, which is realized by the following steps:
s1, fusing the human mesenchymal stem cells cultured in vitro to 85-95%, discarding culture supernatant, adding a plasmid liposome compound, and placing the mixture in a cell culture box for transfection culture;
s2, after the transfection culture is finished, removing the supernatant fluid with the plasmid liposome compound, adding an alpha-MEM culture medium containing the fetal calf serum without the exosome and chloroquine or chloroquine phosphate solution, and placing the mixture in a cell culture box for culture;
s3, collecting the culture supernatant, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the plasmid-carrying exosome.
In S1, the human mesenchymal stem cell may be a primary cell or an immortalized cell.
In the S2, the percentage of the exosome-free fetal calf serum is 8-12%; the concentration of the added chloroquine or chloroquine phosphate solution is 10-50 uM.
In the S3, the time for culturing the cell culture box is 12-48 h.
Compared with the prior art, the plasmid-carrying exosome is obtained by adopting the production method, and the plasmid-encapsulated exosome (POPO-3 fluorescence intensity represents the amount of the plasmid-encapsulated exosome) is effectively obtained, in addition, the method adds a lysosome inhibitor into the transfected cell after the transfection of the mesenchymal stem cell for a specific time by using the plasmid liposome compound transfection technology, so that the efficiency of the mesenchymal stem cell for secreting the plasmid-encapsulated exosome is remarkably increased, namely, the plasmid liposome compound is used for transfecting the mesenchymal stem cell, and then lysosome inhibition is combined, so that the plasmid-carrying exosome can be effectively produced; the exosome can be easily taken up by receptor cells to play the therapeutic effect of plasmids, and the exosome has no obvious immunological rejection reaction. The exosome carrying the plasmid can be widely used as a novel medicament for treating various diseases.
Drawings
FIG. 1 is a graph showing the fluorescence intensity of a plasmid loaded without addition of a lysosomal inhibitor in the present example;
FIG. 2 is a bar graph showing the fluorescence intensity of the plasmid loaded without the addition of the lysosomal inhibitor in the present example;
FIG. 3 is a graph showing the fluorescence intensity of plasmid-loaded exosomes obtained in example 3 of the present invention after addition of a lysosomal inhibitor;
FIG. 4 is a graph showing the fluorescence intensity of plasmid-loaded exosomes obtained in example 3 of the present invention when cultured for 24 h;
FIG. 5 is a graph showing the fluorescence intensity of plasmid-loaded exosomes obtained in example 3 of the present invention when cultured for 48 hours.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It is to be noted that all the components used in the following examples are commercially or self-made.
In addition, the plasmid-liposome complexes described in the following examples were obtained by:
1) respectively mixing 2.5-19.5 ug of plasmid, 5-39 ul of P3000 and 0.25-1.95 ml of Opti-MEM to obtain a first mixture;
2) respectively taking 5-39 ul of Lipo3000 and 0.25-1.95 ml of Opti-MEM, and uniformly mixing to obtain a second mixture;
3) and combining the first mixture and the second mixture, gently mixing uniformly, and standing for 10-30 min to obtain the plasmid-liposome complex.
The method for producing the plasmid-carried exosome provided by the embodiment of the invention is realized by the following steps:
s1, fusing the human-derived mesenchymal stem cells cultured in vitro to 85-95%, discarding culture supernatant, and adding 1-10 ml of Opti-MEM to obtain the human-derived mesenchymal stem cells containing the Opti-MEM cell culture solution. Adding the plasmid liposome complex into the human-derived mesenchymal stem cells containing the Opti-MEM cell culture solution, and placing the cells into a cell culture box for transfection culture for 4-8 h;
s2, after the transfection culture is completed, removing the supernatant with the plasmid liposome compound, adding an alpha-MEM culture medium containing 8-12% of an exosome-free fetal calf serum and chloroquine or chloroquine phosphate solution with the concentration of 20-30 uM, shaking uniformly in a cross manner, and placing in a cell culture box for culture for 12-48h respectively;
s3, collecting the culture supernatant, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the plasmid-carrying exosome.
The following are specific examples
EXAMPLE 1
The plasmid liposome complex provided by the embodiment 1 of the invention is obtained by the following steps:
1) 19.5ug of plasmid, 39ul of P3000 and 1.95ml of Opti-MEM were mixed to obtain a first mixture; 2) respectively taking 39ul of Lipo3000 and 1.95ml of Opti-MEM, and uniformly mixing to obtain a second mixture; 3) and combining the first mixture and the second mixture, gently mixing uniformly, and standing for 20min to obtain the plasmid-liposome complex.
EXAMPLE 2
The plasmid liposome complex provided by the embodiment 2 of the invention is obtained by the following steps:
1) respectively taking 2.5ug of plasmid, 5ul of P3000 and 0.25ml of Opti-MEM to mix to obtain a first mixture; 2) respectively taking 5ul of Lipo3000 and 0.25ml of Opti-MEM to be uniformly mixed to obtain a second mixture; 3) and combining the first mixture and the second mixture, gently mixing uniformly, and standing for 10min to obtain the plasmid-liposome complex.
EXAMPLE 3
The method for producing the plasmid-carrying exosome provided by the embodiment 3 of the invention is implemented by the following steps:
s1, transfection method: culturing human mesenchymal stem cells outside a T75 culture flask body until the fusion of the human mesenchymal stem cells reaches 90%, discarding culture supernatant, adding 6ml of Opti-MEM, and culturing in a cell culture box to obtain the human mesenchymal stem cells containing the Opti-MEM cell culture solution. Adding the plasmid liposome compound obtained in the embodiment 1 of the invention into human-derived mesenchymal stem cells containing an Opti-MEM cell culture solution, shaking uniformly, and placing in a cell culture box for transfection culture for 6 h;
s2, lysosomal inhibition: after transfection, the supernatant with plasmid-liposome complex was removed, α -MEM medium containing 10% of exosome-free fetal bovine serum and chloroquine or chloroquine phosphate solution at a concentration of 25uM were added, shaken up, and placed in a cell culture chamber for 24h and 48h, respectively.
S3, purifying exosome: collecting the supernatant after 24h and 48h of culture, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the exosome carrying the plasmid.
Example 4
The method for producing the plasmid-carrying exosome provided by the embodiment 4 of the invention is implemented by the following steps:
s1, transfection method: culturing human mesenchymal stem cells outside a T75 culture flask body until the human mesenchymal stem cells are fused to 85%, discarding culture supernatant, adding 6ml of Opti-MEM, and culturing in cell culture to obtain the human mesenchymal stem cells containing the Opti-MEM cell culture solution. Adding the plasmid liposome compound obtained in the embodiment 1 of the invention into human-derived mesenchymal stem cells containing an Opti-MEM cell culture solution, shaking uniformly, and placing in a cell culture box for transfection culture for 6 h;
s2, lysosomal inhibition: after transfection, the supernatant with plasmid-liposome complex was removed, α -MEM medium containing 10% of exosome-free fetal bovine serum and chloroquine or chloroquine phosphate solution at a concentration of 25uM were added, shaken up, and placed in a cell culture chamber for 24h and 48h, respectively.
S3, purifying exosome: collecting the supernatant after 24h and 48h of culture, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the exosome carrying the plasmid.
Example 5
The method for producing the plasmid-carrying exosome provided by the embodiment 5 of the invention is implemented by the following steps:
s1, transfection method: culturing human mesenchymal stem cells outside a T75 culture flask body until the fusion of the human mesenchymal stem cells reaches 95%, discarding culture supernatant, adding 6ml of Opti-MEM, and culturing in cell culture to obtain the human mesenchymal stem cells containing the Opti-MEM cell culture solution. Adding the plasmid liposome compound obtained in the embodiment 1 of the invention into human mesenchymal stem cells containing an Opti-MEM cell culture solution for culture, shaking uniformly, and placing the mixture into a cell culture box for transfection culture for 6 hours;
s2, lysosomal inhibition: after transfection, the supernatant with plasmid-liposome complex was removed, α -MEM medium containing 10% of exosome-free fetal bovine serum and chloroquine or chloroquine phosphate solution at a concentration of 25uM were added, shaken up, and placed in a cell culture chamber for 24h and 48h, respectively.
S3, purifying exosome: collecting the supernatant after 24h and 48h of culture, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the exosome carrying the plasmid.
EXAMPLE 6
The method for producing the plasmid-carrying exosome provided by the embodiment 6 of the invention is implemented by the following steps:
s1, transfection method: adopting a six-hole plate for in vitro culture of the human-derived mesenchymal stem cells to fuse to 90 percent, discarding culture supernatant, adding 1ml of Opti-MEM, and culturing in cell culture to obtain the human-derived mesenchymal stem cells containing the Opti-MEM cell culture solution. Adding the plasmid liposome compound obtained in the embodiment 2 of the invention into human-derived mesenchymal stem cells containing an Opti-MEM cell culture solution, shaking uniformly, and placing in a cell culture box for transfection culture for 6 h;
s2, lysosomal inhibition: after transfection, the supernatant with plasmid-liposome complex was removed, α -MEM medium containing 10% of exosome-free fetal bovine serum and chloroquine or chloroquine phosphate solution at a concentration of 25uM were added, shaken up, and placed in a cell culture chamber for 24h and 48h, respectively.
S3, purifying exosome: collecting the supernatant after 24h and 48h of culture, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the exosome carrying the plasmid.
In order to verify the effect of plasmid-carrying exosomes obtained by the method for producing plasmid-carrying exosomes of the present invention, the plasmid-carrying exosomes obtained in example 4 of the present invention were tested for fluorescence properties, and the plasmid-carrying exosomes without added lysosomal inhibitor were also tested for fluorescence properties for comparison, and the specific test results are shown in fig. 1 to fig. 4 below:
FIGS. 1 and 2 are graphs showing the fluorescence properties of the loaded plasmid without addition of the lysosomal inhibitor; FIGS. 3, 4 and 5 are graphs showing the fluorescence properties of the loaded plasmid with added lysosomal inhibitor;
as can be seen by comparison, the fluorescence properties of the plasmid loaded with the lysosomal inhibitor are significantly better than those of the plasmid loaded without the lysosomal inhibitor.
In conclusion, the plasmid-carrying exosome is obtained by the production method, and the plasmid-carrying exosome is effectively obtained, in addition, the method provided by the invention remarkably increases the efficiency of the mesenchymal stem cell for secreting the plasmid-encapsulated exosome by adding the lysosome inhibitor into the transfected cell after the transfection of the mesenchymal stem cell for a specific time by using the plasmid liposome complex transfection technology, namely, the plasmid-carrying exosome can be effectively produced by transfecting the mesenchymal stem cell by using the plasmid liposome complex and combining lysosome inhibition; the exosome can be easily taken up by receptor cells to play the therapeutic effect of plasmids, and the exosome has no obvious immunological rejection reaction. The exosome carrying the plasmid can be widely used as a novel medicament for treating various diseases.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (4)
1. A method for producing a plasmid-carrying exosome, comprising the steps of:
s1, fusing the human mesenchymal stem cells cultured in vitro to 85-95%, discarding culture supernatant, adding a plasmid liposome compound, and placing the mixture in a cell culture box for transfection culture;
s2, after the transfection culture is finished, removing the supernatant fluid with the plasmid liposome compound, adding an alpha-MEM culture medium containing the fetal calf serum without the exosome and chloroquine or chloroquine phosphate solution, and placing the mixture in a cell culture box for culture;
s3, collecting the culture supernatant, and separating and purifying by using an ultracentrifugation or exosome extraction kit to obtain the plasmid-carrying exosome.
2. The method for producing plasmid-carrying exosomes according to claim 1, wherein in S1, the human mesenchymal stem cells used may be primary cells or immortalized cells.
3. The method for producing a plasmid-carrying exosome according to claim 2, wherein the percentage of the exosome-free fetal bovine serum in the S2 is 8-12%; the concentration of the added chloroquine or chloroquine phosphate solution is 10-50 uM.
4. The method for producing a plasmid-harbored exosome according to claim 3, wherein in S2, the cell culture chamber is cultured for 12 to 48 hours.
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| CN115141804A (en) * | 2022-09-05 | 2022-10-04 | 南京鼓楼医院 | Neuron transfection method based on fat exosomes and application thereof |
| CN115141804B (en) * | 2022-09-05 | 2023-01-20 | 南京鼓楼医院 | Neuron transfection method based on fat exosomes and application thereof |
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