CN112553149A - Preparation method of exosome - Google Patents
Preparation method of exosome Download PDFInfo
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- CN112553149A CN112553149A CN201910909054.2A CN201910909054A CN112553149A CN 112553149 A CN112553149 A CN 112553149A CN 201910909054 A CN201910909054 A CN 201910909054A CN 112553149 A CN112553149 A CN 112553149A
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- exosome
- supernatant
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- exosomes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a preparation method of exosome, which comprises the following steps: s1 preparation of umbilical cord mesenchymal stem cell supernatant, S2 separation and storage of umbilical cord mesenchymal stem cell exosome, and S3 exosome extraction.
Description
Technical Field
The invention belongs to the field of exosome preparation, and particularly relates to a preparation method of an exosome.
Background
The exosome is a small membrane vesicle (30-150nm) containing complex RNA and protein, and nowadays, the exosome refers to a disc-shaped vesicle with the diameter of 40-100nm, various cells can secrete the exosome under normal and pathological states, the exosome mainly comes from a multi-vesicle body formed by invagination of intracellular lysosome particles, and the multi-vesicle body is released into an extracellular matrix after fusion of an outer membrane and a cell membrane of the multi-vesicle body.
Disclosure of Invention
The present invention aims to provide a method for preparing exosome, so as to solve the problems proposed in the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of exosome comprises the following steps:
s1, preparing the supernatant of the umbilical cord mesenchymal stem cells, namely adding a culture medium without exosome serum into the umbilical cord mesenchymal stem cells, diluting the culture medium into cell suspension, inoculating the cell suspension into a cell culture bottle for culture, and collecting the cultured supernatant;
s2, separating and preserving umbilical cord mesenchymal stem cell exosomes, treating the supernatant obtained in the step S1, firstly, filtering cell impurities and dead cells in the supernatant, filtering by using an aseptic filter membrane, subpackaging the obtained exosome precipitate in an aseptic centrifuge tube, preserving for later use, then diluting the exosome precipitate into cell suspension, inoculating the cell suspension in 25cm3The cell culture flask of (1) was placed at 45 ℃ in 6% CO2Collecting supernatant after cell culture when the culture is carried out for 40-80h in an incubator with saturated humidity, collecting the supernatant to more than 250ml, and extracting exosome;
s3, extracting exosomes, firstly, centrifuging the liquid obtained in the step S2, centrifuging the supernatant, transferring the supernatant, removing dead cells in the supernatant, then filtering the supernatant through a sterile filter membrane of 0.22 mu m to remove impurities in the supernatant, secondly, ultracentrifuging the obtained supernatant to precipitate exosomes, removing the supernatant, adding 500 mu l of PBS to dissolve and collect exosome precipitates, finally filtering the exosome precipitates through the sterile filter membrane of 0.22 mu m to obtain concentrated exosome concentrated solution, adding sodium lactate, sodium chloride and potassium chloride to dissolve the concentrated exosome concentrated solution to obtain exosome salt solution, adding the exosome extracting solution into the exosome salt solution to culture, ultracentrifuging the cultured solution at 12000g at 4 ℃ for 70min to precipitate exosomes, and subpackaging the obtained exosome precipitates in a sterile centrifuge tube for later use at-80 ℃.
Preferably, in step S2, when the sample concentration is too high, dilution is performed by adding an equal volume of PBS buffer.
Preferably, in step S1, the culture medium without exosome serum is added into umbilical cord mesenchymal stem cells, and the umbilical cord mesenchymal stem cells are cultured for 24-48h, and the supernatant is collected when the cell fusion degree reaches about 80% -95%.
Preferably, in step S3, the supernatant is first centrifuged at 4 ℃ for 10min at 2000g to remove dead cells, and then the exosomes are precipitated by ultracentrifugation at 4 ℃ for 60min at 10000 g.
Preferably, in step S3, the pore size of the filter used for filtering and removing the vesicles is 0.20 μm to 0.24 μm, and 0.22 μm may be selected.
Preferably, in step S3, the centrifugation is performed at 1800g to 2200g at a low temperature of 2 ℃ to 6 ℃.
Compared with the prior art, the invention has the beneficial effects that: compared with the preparation method of the conventional exosome, the exosome derived from the umbilical cord mesenchymal stem cells has stronger immunoregulation function, can be used for treating autoimmune diseases such as lupus erythematosus and scleroderma, reduces the immune rejection reaction after cell or organ transplantation, improves the success rate of cell or organ transplantation, and increases the purity of the exosome by adding a mixed salt solution of sodium lactate, sodium chloride and potassium chloride into the obtained exosome concentrated solution for culture and then extracting.
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.
A preparation method of exosome comprises the following steps:
s1, preparing the supernatant of the umbilical cord mesenchymal stem cells, namely adding a culture medium without exosome serum into the umbilical cord mesenchymal stem cells, diluting the culture medium into cell suspension, inoculating the cell suspension into a cell culture bottle for culture, and collecting the cultured supernatant;
s2, separating and preserving umbilical cord mesenchymal stem cell exosomes, treating the supernatant obtained in the step S1, firstly, filtering cell impurities and dead cells in the supernatant, filtering by using a sterile filter membrane, and obtaining the exosomesThe precipitate is subpackaged in a sterile centrifuge tube, stored for later use, and then diluted into cell suspension to be inoculated in 25cm3The cell culture flask of (1) was placed at 45 ℃ in 6% CO2Collecting supernatant after cell culture when the culture is carried out for 40-80h in an incubator with saturated humidity, collecting the supernatant to more than 250ml, and extracting exosome;
s3, extracting exosomes, firstly, centrifuging the liquid obtained in the step S2, centrifuging the supernatant, transferring the supernatant, removing dead cells in the supernatant, then filtering the supernatant through a sterile filter membrane of 0.22 mu m to remove impurities in the supernatant, secondly, ultracentrifuging the obtained supernatant to precipitate exosomes, removing the supernatant, adding 500 mu l of PBS to dissolve and collect exosome precipitates, finally filtering the exosome precipitates through the sterile filter membrane of 0.22 mu m to obtain concentrated exosome concentrated solution, adding sodium lactate, sodium chloride and potassium chloride to dissolve the concentrated exosome concentrated solution to obtain exosome salt solution, adding the exosome extracting solution into the exosome salt solution to culture, ultracentrifuging the cultured solution at 12000g at 4 ℃ for 70min to precipitate exosomes, and subpackaging the obtained exosome precipitates in a sterile centrifuge tube for later use at-80 ℃.
Specifically, in step S2, when the sample concentration is too high, dilution is performed by adding an equal volume of PBS buffer.
Specifically, in step S1, adding a culture medium without exosome serum into the umbilical cord mesenchymal stem cells, culturing for 24-48h, and collecting supernatant when the cell fusion degree reaches about 80% -95%.
Specifically, in step S3, the supernatant was centrifuged at 4 ℃ for 10min to remove dead cells at 2000g, and then the exosome was precipitated by ultracentrifugation at 4 ℃ for 60min at 10000 g.
Specifically, in step S3, the pore size of the filter used for filtering out the vesicles is 0.20 to 0.24 μm, and 0.22 μm may be selected.
Specifically, in step S3, the centrifugation is performed at 1800g to 2200g at a low temperature of 2 ℃ to 6 ℃.
Compared with the conventional preparation method of the exosome, the exosome derived from the umbilical cord mesenchymal stem cells has stronger immunoregulation function, can be used for treating autoimmune diseases such as lupus erythematosus and scleroderma, reduces immune rejection after cell or organ transplantation, improves the success rate of cell or organ transplantation, and increases the purity of the exosome by adding sodium lactate, sodium chloride and potassium chloride salt solution into the obtained exosome concentrated solution for culture and then extracting.
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 person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
1. A preparation method of exosome is characterized by comprising the following steps:
s1, preparing the supernatant of the umbilical cord mesenchymal stem cells, namely adding a culture medium without exosome serum into the umbilical cord mesenchymal stem cells, diluting the culture medium into cell suspension, inoculating the cell suspension into a cell culture bottle for culture, and collecting the cultured supernatant;
s2, separating and preserving umbilical cord mesenchymal stem cell exosomes, treating the supernatant obtained in the step S1, firstly, filtering cell impurities and dead cells in the supernatant, filtering by using an aseptic filter membrane, subpackaging the obtained exosome precipitate in an aseptic centrifuge tube, preserving for later use, then diluting the exosome precipitate into cell suspension, inoculating the cell suspension in 25cm3The cell culture flask of (1) was placed at 45 ℃ in 6% CO2Collecting supernatant after cell culture when the culture is carried out for 40-80h in an incubator with saturated humidity, collecting the supernatant to more than 250ml, and extracting exosome;
s3, extracting exosomes, firstly, centrifuging the liquid obtained in the step S2, centrifuging the supernatant, transferring the supernatant, removing dead cells in the supernatant, then filtering the supernatant through a sterile filter membrane of 0.22 mu m to remove impurities in the supernatant, secondly, ultracentrifuging the obtained supernatant to precipitate exosomes, removing the supernatant, adding 500 mu l of PBS to dissolve and collect exosome precipitates, finally filtering the exosome precipitates through the sterile filter membrane of 0.22 mu m to obtain concentrated exosome concentrated solution, adding sodium lactate, sodium chloride and potassium chloride to dissolve the concentrated exosome concentrated solution to obtain exosome salt solution, adding the exosome extracting solution into the exosome salt solution to culture, ultracentrifuging the cultured solution at 12000g at 4 ℃ for 70min to precipitate exosomes, and subpackaging the obtained exosome precipitates in a sterile centrifuge tube for later use at-80 ℃.
2. A method of producing exosomes according to claim 1, characterized in that: in step S2, when the sample concentration is too high, dilution is performed by adding an equal volume of PBS buffer.
3. A method of producing exosomes according to claim 1, characterized in that: in step S1, adding a culture medium without exosome serum into the umbilical cord mesenchymal stem cells, culturing for 24-48h, and collecting supernatant when the cell fusion degree reaches about 80% -95%.
4. A method of producing exosomes according to claim 1, characterized in that: in step S3, the supernatant was first centrifuged at 4 ℃ for 10min at 2000g to remove dead cells, and then the exosomes were precipitated by ultracentrifugation at 4 ℃ for 60min at 10000 g.
5. A method of producing exosomes according to claim 1, characterized in that: in step S3, the pore size of the filter used for filtering and removing the vesicles is 0.20 to 0.24. mu.m, and 0.22. mu.m may be selected.
6. A method of producing exosomes according to claim 1, characterized in that: in step S3, the mixture is centrifuged at 1800g to 2200g at a low temperature of 2 ℃ to 6 ℃.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113502256A (en) * | 2021-06-11 | 2021-10-15 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Extraction method of sterile exosomes |
| CN113577823A (en) * | 2021-07-26 | 2021-11-02 | 云南聚云基因工程有限公司 | Glucan gel chromatographic column and method for separating umbilical cord blood mesenchymal stem cell exosome |
| CN113801894A (en) * | 2021-09-17 | 2021-12-17 | 香港大学深圳医院 | Production method of plasmid-carried exosome |
| CN114042087A (en) * | 2021-09-30 | 2022-02-15 | 上海市同济医院 | Application of human umbilical cord mesenchymal stem cell exosome in preparation of medicine for treating scleroderma |
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Patent Citations (4)
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| CN108642004A (en) * | 2018-05-17 | 2018-10-12 | 广东芙金干细胞再生医学有限公司 | A kind of preparation method of excretion body and the stem cell proliferating agent containing excretion body |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113502256A (en) * | 2021-06-11 | 2021-10-15 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Extraction method of sterile exosomes |
| CN113502256B (en) * | 2021-06-11 | 2023-09-19 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Extraction method of sterile exosomes |
| CN113577823A (en) * | 2021-07-26 | 2021-11-02 | 云南聚云基因工程有限公司 | Glucan gel chromatographic column and method for separating umbilical cord blood mesenchymal stem cell exosome |
| CN113801894A (en) * | 2021-09-17 | 2021-12-17 | 香港大学深圳医院 | Production method of plasmid-carried exosome |
| CN113801894B (en) * | 2021-09-17 | 2023-08-22 | 香港大学深圳医院 | Production method of exosomes carrying plasmids |
| CN114042087A (en) * | 2021-09-30 | 2022-02-15 | 上海市同济医院 | Application of human umbilical cord mesenchymal stem cell exosome in preparation of medicine for treating scleroderma |
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Application publication date: 20210326 |