CN117603907A - Extraction method of mesenchymal stem cell exosomes - Google Patents
Extraction method of mesenchymal stem cell exosomes Download PDFInfo
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- CN117603907A CN117603907A CN202311577937.0A CN202311577937A CN117603907A CN 117603907 A CN117603907 A CN 117603907A CN 202311577937 A CN202311577937 A CN 202311577937A CN 117603907 A CN117603907 A CN 117603907A
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 68
- 210000001808 exosome Anatomy 0.000 title claims abstract description 60
- 238000000605 extraction Methods 0.000 title claims abstract description 26
- 238000005199 ultracentrifugation Methods 0.000 claims abstract description 38
- 239000012228 culture supernatant Substances 0.000 claims abstract description 37
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 27
- 229930006000 Sucrose Natural products 0.000 claims abstract description 27
- 238000005119 centrifugation Methods 0.000 claims abstract description 27
- 239000005720 sucrose Substances 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000006228 supernatant Substances 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 12
- 210000003954 umbilical cord Anatomy 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 210000002826 placenta Anatomy 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 abstract description 10
- 230000001066 destructive effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 12
- 239000002245 particle Substances 0.000 description 10
- 239000000725 suspension Substances 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 102000004895 Lipoproteins Human genes 0.000 description 3
- 108090001030 Lipoproteins Proteins 0.000 description 3
- 238000001085 differential centrifugation Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- CPKVUHPKYQGHMW-UHFFFAOYSA-N 1-ethenylpyrrolidin-2-one;molecular iodine Chemical compound II.C=CN1CCCC1=O CPKVUHPKYQGHMW-UHFFFAOYSA-N 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 229920000153 Povidone-iodine Polymers 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
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- 229960001621 povidone-iodine Drugs 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
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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
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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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- 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
- C12N2509/10—Mechanical dissociation
Abstract
The invention provides a method for extracting mesenchymal stem cell exosomes, and relates to the technical field of extraction of mesenchymal stem cell exosomes. The method comprises the following steps: collecting culture supernatant of the mesenchymal stem cells, performing pretreatment, preparing sucrose water pad, and mixing the sucrose water pad with the treated culture supernatant according to the following ratio of 1:7-10, and performing two times of ultracentrifugation, removing supernatant after the second time of ultracentrifugation, and re-suspending the bottom precipitated mesenchymal stem cell exosomes with a certain amount of phosphate buffer solution PBS to complete collection. The recovery amount of exosomes extracted by the method is increased, the recovery efficiency is improved, the use amount of raw materials is reduced, the production efficiency is higher, and the overall extraction cost is reduced; the exosomes are not easy to damage and have good integrity in the extraction process, so that destructive loss caused by recovering the exosomes by a differential ultracentrifugation method is reduced; and the culture supernatant is subjected to three times of centrifugation pretreatment, so that the extraction purity of exosomes is improved.
Description
Technical Field
The invention relates to the technical field of extraction of mesenchymal stem cell exosomes, in particular to an extraction method of mesenchymal stem cell exosomes.
Background
The differential centrifugation adopts a method of gradually increasing the centrifugal speed or alternately centrifuging at a low speed and a high speed to separate particles with different sedimentation speeds in batches under different centrifugal speeds and different centrifugal times. Generally, the low centrifugal speed enables larger particles to settle, small particles are still suspended in the supernatant, the suspension is centrifuged at the high centrifugal speed after the sediment is collected, and the small particles are settled, so that the purpose of separating particles with different sizes is achieved.
The conventional three-time differential centrifugation method is often adopted in exosome separation, so that time and labor are wasted, good purity cannot be achieved, and the exosome separation is easy to be polluted by other lipoproteins and the like.
Disclosure of Invention
The invention provides a method for extracting mesenchymal stem cell exosomes, which overcomes the defects existing in the prior art.
The specific technical scheme is that the extraction method of mesenchymal stem cell exosomes comprises the following steps:
s1, collecting culture supernatant for culturing mesenchymal stem cells, performing pretreatment,
s2, preparing sucrose water pad liquid with the solute mass ratio of 30 percent,
s3, first ultracentrifugation: mixing the sucrose water pad prepared in the step S2 with the culture supernatant treated in the step S1 according to the following ratio of 1:7-10, and performing first ultracentrifugation,
s4, second ultracentrifugation: after the first ultracentrifugation is completed, collecting a light white fog layer between sucrose water cushion liquid and culture supernatant into a new centrifuge tube, adding phosphate buffer PBS to fill the centrifuge tube to a constant volume, performing the second ultracentrifugation,
s5, after the second ultracentrifugation is finished, removing supernatant, and re-suspending mesenchymal stem cell exosomes precipitated at the bottom by using a certain amount of phosphate buffer solution PBS to finish collection.
Further, in step S1, a culture supernatant of umbilical cord/placenta mesenchymal stem cells cultured using a complete medium of mesenchymal stem cells without exosomes is collected.
Further, the culture supernatant of the collected mesenchymal stem cells was subjected to three centrifugation pretreatment.
Further, the centrifugation pretreatment was to sequentially subject the collected culture supernatant to centrifugation of 300g for 10 minutes to collect the supernatant, centrifugation of 1000g for 10 minutes to collect the supernatant, and centrifugation of 10000g for 30 minutes to collect the supernatant.
Further, the first ultracentrifugation condition was centrifugation at 100.000g for 90 minutes at 4 ℃.
Further, the second ultracentrifugation condition was centrifugation at 100.000g for 90 minutes at 4 ℃.
Further, the sucrose water pad liquid is prepared from sucrose: the solvent mass ratio is 30 percent.
Further, in step S3, the ratio of the sucrose water pad prepared in step S2 to the culture supernatant treated in step S1 is 1:10.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following technical advantages:
1. by optimizing the extraction process, namely adopting sucrose water cushion liquid to perform double differential ultracentrifugation on the culture supernatant of the mesenchymal stem cells, the recovery amount of the extracted exosomes is increased, the recovery efficiency is improved, the use amount of raw materials is reduced, the production efficiency is higher, the overall extraction cost is reduced,
2. the extraction method in the application carries out differential ultracentrifugation twice, the exosomes are not easy to be destroyed in the extraction process, the integrity is good, the destructive loss caused by recovering the exosomes by the differential ultracentrifugation method is reduced,
3. the culture supernatant of the collected mesenchymal stem cells is subjected to three-time centrifugation pretreatment to remove residual cells, dead cells and cell fragments, thereby being beneficial to improving the extraction purity of exosomes, simultaneously leading the extraction process to be more visual, reducing the risk of reducing the recovery quantity caused by misoperation,
4. the collected culture supernatant of umbilical cord/placenta mesenchymal stem cells cultured by using a complete culture medium without exosomes reduces the pollution of other lipoproteins and the like, and improves the extraction purity of exosomes.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is a white light diagram of the culture of mesenchymal stem cells of the present invention,
FIG. 2 is a flow chart of the identification of mesenchymal stem cells of the present invention,
FIG. 3 is an electron micrograph of an intermediate mesenchymal stem cell exosome of the present invention,
FIG. 4 is a graph showing the particle size distribution of the mesenchymal stem cell exosomes of the present invention,
FIG. 5 is a phenotypic nanoflow of the intermediate mesenchymal stem cell exosomes of the present invention,
FIG. 6 is a graph showing the comparison of protein content of mesenchymal stem cell exosomes extracted by different methods according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the embodiments and the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. The exemplary embodiments of the present invention and the descriptions thereof are used herein to explain the present invention, but are not intended to limit the invention.
The invention is described below with reference to the accompanying drawings:
the extraction method of the mesenchymal stem cell exosome comprises the following steps:
s1, collecting culture supernatant for culturing mesenchymal stem cells, performing pretreatment,
s2, preparing sucrose water pad liquid with the solute mass ratio of 30 percent,
s3, first ultracentrifugation: mixing the sucrose water pad prepared in the step S2 with the culture supernatant treated in the step S1 according to the following ratio of 1:7-10, and performing first ultracentrifugation,
s4, second ultracentrifugation: after the first ultracentrifugation is completed, collecting a light white fog layer between sucrose water cushion liquid and culture supernatant into a new centrifuge tube, adding phosphate buffer PBS to fill the centrifuge tube to a constant volume, performing the second ultracentrifugation,
s5, after the second ultracentrifugation is finished, removing supernatant, and re-suspending mesenchymal stem cell exosomes precipitated at the bottom by using a certain amount of phosphate buffer solution PBS to finish collection.
In one embodiment, in step S1, the culture supernatant of umbilical cord/placenta mesenchymal stem cells cultured with the exosome-free mesenchymal stem cell complete medium is collected.
In one embodiment, the culture supernatant of the collected mesenchymal stem cells is subjected to three centrifugation pretreatment. And the culture supernatant of the collected mesenchymal stem cells is subjected to three-time centrifugation pretreatment, so that residual cells, dead cells and cell fragments are removed, the extraction purity of exosomes is improved, the extraction process is more visual, and the risk of reduction of recovery caused by misoperation is reduced.
In one embodiment, the centrifugation pretreatment is to sequentially subject the collected culture supernatant to 300g centrifugation for 10 minutes to collect the supernatant, 1000g centrifugation for 10 minutes to collect the supernatant, and 10000g centrifugation for 30 minutes to collect the supernatant.
In one embodiment, the first ultracentrifugation condition is centrifugation at 100.000g for 90 minutes at 4 ℃.
In one embodiment, the second ultracentrifugation condition is centrifugation at 100.000g for 90 minutes at 4 ℃.
In one embodiment, the sucrose water pad is prepared from sucrose: the solvent mass ratio is 30 percent.
In one example, in step S3, the sucrose water pad prepared in step S2 is added to the centrifuge tube at a ratio of 1:10 to the culture supernatant treated in step S1.
Examples:
s101, obtaining a mesenchymal stem cell culture solution: taking fresh healthy human umbilical cord/placenta, sterilizing and washing the tissue with povidone iodine, removing the required umbilical cord/placenta tissue with scissors forceps, fully cutting the tissue, adding collagenase enzyme to eliminate into single primary tissue cells, adding mesenchymal stem cells, completely culturing, suspending at 37deg.C, and 5% CO 2 Culturing in an incubator; after 3-5 days of cell culture, mesenchymal stem cells with a fine spindle shape can be observed under a white light chart, andand the coverage area of the cells can reach 80%, the primary cells can be subjected to 0.125% trypsin digestion subculture according to the understanding of fig. 1, meanwhile, the mesenchymal stem cells are subjected to surface marker identification purity, and the mesenchymal stem cell complete culture medium added in the subsequent subculture can be collected for extraction of mesenchymal stem cell exosomes after the culture is completed according to the understanding of fig. 2.
S102, pretreatment of a mesenchymal stem cell culture supernatant: the collected culture supernatant was subjected to centrifugation at 300g for 10 minutes to collect the supernatant, centrifugation at 1000g for 10 minutes to collect the supernatant, and centrifugation at 10,000g for 30 minutes to collect the supernatant, and the residual cells, dead cells and cell debris were removed.
S2, preparing sucrose water pad liquid: according to sucrose: the mass ratio of the solvent is 30% to prepare the sucrose water pad liquid.
S3, 80ml of culture supernatant fluid which is subjected to pretreatment and the prepared sucrose water pad fluid are mixed according to the volume ratio of 10:1, into a centrifuge tube, centrifuging at 100.000g for 90 min at 4 ℃,
s4, removing the supernatant at the uppermost layer, collecting a light white fog layer between the water cushion liquid and the culture supernatant to a new centrifuge tube, adding PBS to fill the centrifuge tube to a constant volume, centrifuging for 90 minutes at the temperature of 100.000g and 4 ℃,
s5, removing supernatant fluid, and re-suspending the obtained precipitate by using a proper amount of PBS to obtain the required mesenchymal stem cell exosome suspension.
The extraction step of double differential ultracentrifugation is carried out on the culture supernatant of the mesenchymal stem cells by adopting the sucrose water pad in the application. In the step S101, the mesenchymal stem cells are cultured by using a complete medium of the mesenchymal stem cells without exosomes, so that the pollution of other lipoproteins and the like can be reduced, and the extraction purity of exosomes can be improved.
Comparative example:
the mesenchymal stem cell culture medium was obtained and the mesenchymal stem cell culture supernatant was pretreated as in example 1. Adding 80ml of culture supernatant after pretreatment into a centrifuge tube, centrifuging for 90 minutes at the temperature of 100.000g and 4 ℃, removing supernatant, collecting precipitate, combining the precipitate with a new centrifuge tube, adding PBS to fill the centrifuge tube to a constant volume, centrifuging for 90 minutes at the temperature of 100.000g and 4 ℃, removing supernatant, and re-suspending the obtained precipitate with a proper amount of PBS to obtain the required mesenchymal stem cell exosome suspension.
Identification of mesenchymal stem cell exosomes: taking the mesenchymal stem cell exosome suspension in the embodiment 1, adopting a transmission electron microscope TEM, a nanometer particle size tracker NTA and a nanometer flow meter NANO to identify the surface morphology and the particle size distribution of the mesenchymal stem cell exosome, wherein the surface morphology and the particle size distribution of the mesenchymal stem cell exosome obtained by identification are shown in figures 3 and 4-5, the morphology of the mesenchymal stem cell exosome is tea-tray-shaped, and the mesenchymal stem cell exosome is intensively distributed near 100nm, so that the morphology and the particle size of the mesenchymal stem cell exosome are both in accordance with the standards of exosome;
the same culture supernatant amount was further subjected to comparison of exosome recovery of mesenchymal stem cells extracted by the conventional ultracentrifugation set and the new ultracentrifugation set of the comparative example: the method comprises the steps of taking two groups of mesenchymal stem cell exosome suspensions, firstly using RIPA lysate to lyse exosome membrane proteins, adopting BCA method to measure total protein content and protein standard curve after the lysis, and detecting to obtain the total protein content of the mesenchymal stem cell exosome of the two groups, wherein the total protein content of the exosome extracted by the novel ultracentrifugation group is 2.7 times of that of the conventional ultracentrifugation group, as shown in figure 6, which shows that the extraction method in the application can obviously improve the recovery amount of the mesenchymal stem cell exosome. The sucrose water pad liquid is adopted to carry out double differential ultracentrifugation on the culture supernatant of the mesenchymal stem cells, the exosomes are not easy to damage in the extraction process, the integrity is good, the destructive loss caused by recovering the exosomes by a differential ultracentrifugation method is reduced, and the recovery amount of the extracted exosomes is increased. Compared with the conventional three-time differential centrifugation method, the novel ultracentrifugation method reduces the consumption of raw materials while improving the recovery efficiency, has higher production efficiency and reduces the overall extraction cost.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the equivalent embodiments using the technical disclosure described above. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (8)
1. The extraction method of the mesenchymal stem cell exosome is characterized by comprising the following steps:
s1, collecting culture supernatant for culturing mesenchymal stem cells, performing pretreatment,
s2, preparing sucrose water pad liquid with the solute mass ratio of 30 percent,
s3, first ultracentrifugation: mixing the sucrose water pad prepared in the step S2 with the culture supernatant treated in the step S1 according to the following ratio of 1:7-10, and performing first ultracentrifugation,
s4, second ultracentrifugation: after the first ultracentrifugation is completed, collecting a light white fog layer between sucrose water cushion liquid and culture supernatant into a new centrifuge tube, adding phosphate buffer PBS to fill the centrifuge tube to a constant volume, performing the second ultracentrifugation,
s5, after the second ultracentrifugation is finished, removing supernatant, and re-suspending mesenchymal stem cell exosomes precipitated at the bottom by using a certain amount of phosphate buffer solution PBS to finish collection.
2. The method according to claim 1, wherein in step S1, the culture supernatant of umbilical cord/placenta mesenchymal stem cells is collected and cultured using a mesenchymal stem cell complete medium without exosomes.
3. The method for extracting mesenchymal stem cell exosomes according to claim 2, wherein the culture supernatant of the collected mesenchymal stem cells is subjected to three centrifugation pretreatment.
4. The method for extracting mesenchymal stem cell exosomes according to claim 3, wherein the centrifugation pretreatment is to sequentially subject the collected culture supernatant to centrifugation of 300g for 10 minutes, centrifugation of 1000g for 10 minutes and centrifugation of 10000g for 30 minutes.
5. The method for extracting mesenchymal stem cell exosomes according to any one of claims 1-4, wherein the first ultracentrifugation condition is centrifugation at 100.000g at 4 ℃ for 90 minutes.
6. The method for extracting mesenchymal stem cell exosomes according to any one of claims 1-4, wherein the second ultracentrifugation condition is centrifugation at 100.000g at 4 ℃ for 90 minutes.
7. The method for extracting mesenchymal stem cell exosomes according to any one of claims 1-4, wherein the sucrose water pad is prepared from sucrose: the solvent mass ratio is 30 percent.
8. The method according to any one of claims 1 to 4, wherein in step S3, the ratio of the sucrose aqueous gasket fluid prepared in step S2 to the culture supernatant fluid treated in step S1 is 1:10.
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