CN116716288B - Method for improving exosome yield by acoustic wave vibration - Google Patents
Method for improving exosome yield by acoustic wave vibration Download PDFInfo
- Publication number
- CN116716288B CN116716288B CN202310479553.9A CN202310479553A CN116716288B CN 116716288 B CN116716288 B CN 116716288B CN 202310479553 A CN202310479553 A CN 202310479553A CN 116716288 B CN116716288 B CN 116716288B
- Authority
- CN
- China
- Prior art keywords
- frequency
- cells
- amplitude
- exosome
- exosomes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 42
- 210000004027 cell Anatomy 0.000 claims abstract description 83
- 238000004113 cell culture Methods 0.000 claims abstract description 18
- 238000004519 manufacturing process Methods 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 3
- 244000309466 calf Species 0.000 claims abstract description 3
- 239000012091 fetal bovine serum Substances 0.000 claims abstract description 3
- 210000002966 serum Anatomy 0.000 claims abstract description 3
- 239000006228 supernatant Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 239000002953 phosphate buffered saline Substances 0.000 claims description 9
- 230000001640 apoptogenic effect Effects 0.000 claims description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 6
- 230000001965 increasing effect Effects 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- 239000012930 cell culture fluid Substances 0.000 claims description 2
- 230000006872 improvement Effects 0.000 abstract description 3
- 238000004140 cleaning Methods 0.000 abstract description 2
- 238000001262 western blot Methods 0.000 description 8
- 239000002245 particle Substances 0.000 description 7
- 230000003068 static effect Effects 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000000638 stimulation Effects 0.000 description 6
- 230000005540 biological transmission Effects 0.000 description 5
- 238000004627 transmission electron microscopy Methods 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 4
- 210000002487 multivesicular body Anatomy 0.000 description 4
- 230000000284 resting effect Effects 0.000 description 4
- 241000142710 Isla Vista hantavirus Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000000053 physical method Methods 0.000 description 3
- 102100025222 CD63 antigen Human genes 0.000 description 2
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 2
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 2
- 229930191564 Monensin Natural products 0.000 description 2
- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229960005358 monensin Drugs 0.000 description 2
- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- LSLYOANBFKQKPT-DIFFPNOSSA-N 5-[(1r)-1-hydroxy-2-[[(2r)-1-(4-hydroxyphenyl)propan-2-yl]amino]ethyl]benzene-1,3-diol Chemical compound C([C@@H](C)NC[C@H](O)C=1C=C(O)C=C(O)C=1)C1=CC=C(O)C=C1 LSLYOANBFKQKPT-DIFFPNOSSA-N 0.000 description 1
- MPORYQCGWFQFLA-ONPDANIMSA-N 7-[(7s)-7-amino-5-azaspiro[2.4]heptan-5-yl]-8-chloro-6-fluoro-1-[(1r,2s)-2-fluorocyclopropyl]-4-oxoquinoline-3-carboxylic acid;trihydrate Chemical compound O.O.O.C([C@H]1N)N(C=2C(=C3C(C(C(C(O)=O)=CN3[C@H]3[C@H](C3)F)=O)=CC=2F)Cl)CC11CC1.C([C@H]1N)N(C=2C(=C3C(C(C(C(O)=O)=CN3[C@H]3[C@H](C3)F)=O)=CC=2F)Cl)CC11CC1 MPORYQCGWFQFLA-ONPDANIMSA-N 0.000 description 1
- 102100027221 CD81 antigen Human genes 0.000 description 1
- 101710165845 CD81 protein Proteins 0.000 description 1
- 102000034342 Calnexin Human genes 0.000 description 1
- 108010056891 Calnexin Proteins 0.000 description 1
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 1
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- CWRVKFFCRWGWCS-UHFFFAOYSA-N Pentrazole Chemical compound C1CCCCC2=NN=NN21 CWRVKFFCRWGWCS-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- -1 SB218795 Chemical compound 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 229940014425 exodus Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229960001022 fenoterol Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011528 liquid biopsy Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000028161 membrane depolarization Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- NMTBSNPBIGRZBL-UHFFFAOYSA-N nitrefazole Chemical compound CC1=NC([N+]([O-])=O)=CN1C1=CC=C([N+]([O-])=O)C=C1 NMTBSNPBIGRZBL-UHFFFAOYSA-N 0.000 description 1
- 229950008568 nitrefazole Drugs 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 229960005152 pentetrazol Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229960003177 sitafloxacin Drugs 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000008646 thermal stress Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- 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
- C12N5/0693—Tumour cells; Cancer cells
-
- 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
-
- 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
- C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for improving the output of exosomes by using acoustic wave vibration, which comprises the following steps: subculturing the cells; judging whether the cells are attached, if so, discarding the original culture solution, and culturing the cells by using the exosome-free foetus calf serum after cleaning; applying a preset low-frequency sine wave sound wave to the process of culturing cells by adopting exosome-free fetal bovine serum to obtain a cell culture solution; collecting cell culture solution, and centrifuging to obtain exosomes. The method provided by the invention has the advantages that the cell is stimulated for a certain time by adopting the low-frequency sine wave vibration with specific amplitude, so that the exosome yield of the cell is obviously improved, and the problems that the traditional exosome yield improvement method is low in efficiency, high in cost and difficult to meet the production and living needs are solved.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for improving the output of exosomes by using acoustic wave vibration.
Background
The exosomes are extracellular vesicles with diameters ranging from 30 nm to 100nm secreted by almost all types of cells, exist in intracellular multivesicles, participate in the regulation of normal physiological processes, and can also complete the generation and metastasis of tumors by regulating the crosstalk between tumor cells and medium cells. Therefore, exosomes become novel biomarkers in cancer detection. Exosome detection has become an emerging means for cancer diagnosis and clinical detection by virtue of its lack of invasive surgery and in vitro liquid biopsy.
However, one of the major challenges impeding the transformation of exosomes to clinical use is their low yield, failing to achieve a large-scale and cost-effective production regimen that would be required for preclinical and clinical trials. To solve this problem, biological, chemical and physical methods have been explored to increase exosome production. For example, the use of Monensin (MON) to induce changes in intracellular ca2+ or potassium-induced depolarization results in massive entry of ca2+ stimulating exosome release in cells. In addition, the drugs sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol also activate exosome production and release. Physical methods such as thermal stress, hypoxia and irradiation, pH and the like have all been reported to demonstrate that exosome production can be increased. Unfortunately, the above methods all suffer from drawbacks. The addition of inducing agents and drugs can result in cytotoxicity, reduced cell viability, and the introduction of foreign substances that can reduce exosome purity, requiring further purification complicating the procedure. Physical methods with strong stimulation can denature exosome surface proteins, which may directly affect exosome morphology and performance, compromising their diagnostic and screening capabilities. Therefore, the existing method for improving the yield of the exosomes secreted by the cells has the defects of complex operation, severe experimental conditions and large damage to the cells, and is difficult to produce a large amount of exosomes in a short time, so that the actual production needs and engineering application needs can not be met.
Thus, there is a need for a method of improving exosome production by sonic vibration.
Disclosure of Invention
In view of the above problems, the present invention provides a method for improving the output of exosomes by using acoustic wave vibration, so as to solve the problem that the existing method for improving the output of exosomes secreted by cells is difficult to produce a large amount of exosomes in a short time.
In order to solve the technical problem, the specification provides the following technical scheme:
a method for improving the output of exosomes by using acoustic wave vibration, comprising the following steps:
subculturing the cells;
judging whether cells adhere to the wall, if so, discarding the original culture solution; culturing cells with exosome-free foetal calf serum after washing;
applying a preset low-frequency sine wave sound wave to the process of culturing cells by adopting exosome-free bovine serum to obtain a cell culture solution;
Collecting cell culture solution, and centrifuging to obtain exosomes.
Further, the frequency range of the low-frequency sine wave sound wave is 50 Hz-200 Hz, the amplitude range is 20-100 μm, and the application time is 6 h-72 h.
Further, the process of applying the low-frequency sine wave sound wave is sequentially carried out according to the following system:
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 20 μm;
Sound wave 6 h with frequency of 100 Hz-200 Hz and amplitude of 40 μm;
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 60 μm;
Sound wave 12 h with frequency of 100 Hz-200 Hz and amplitude of 80 μm;
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 60 μm;
Sound wave 6 h with frequency of 100 Hz-200 Hz and amplitude of 40 μm;
sound wave 6 h with a frequency of 50 Hz-100 Hz and an amplitude of 20 μm.
Further, the mode of applying the low-frequency sine wave sound wave is as follows:
Placing a speaker under the cell culture vessel;
controlling the continuous playing frequency and amplitude of the loudspeaker to meet the set requirements;
Judging whether the playing time of the loudspeaker reaches a preset time threshold, and if so, collecting the cell culture fluid.
Further, a mobile digital terminal is adopted to control the playing frequency, amplitude and time of the loudspeaker.
Further, the centrifugation process is as follows:
centrifuging the cell culture solution by using a relative centrifugal force of 2000g to obtain a supernatant from which cell fragments and dead cells are removed;
centrifuging the supernatant from which cell debris and dead cells are removed by using a relative centrifugal force of 12000g to obtain a supernatant from which microbubbles or protein aggregates are removed;
Filtering the supernatant from which the microbubbles or protein aggregates are removed by using a 0.22 μm filter to obtain a supernatant from which apoptotic bodies and microbubbles are removed;
and adding the supernatant with the apoptotic bodies and microbubbles removed into an exosome separation instrument for separation to obtain exosomes.
Further, after the exosomes are obtained, the following process is also included:
the exosomes were washed with phosphate buffered saline and then resuspended with phosphate buffered saline.
Further, the cell subculturing conditions were as follows:
cells were placed in an incubator at 37℃and subcultured for 24 hours. .
Further, the process of washing the cells is as follows:
Cells were washed twice with phosphate buffered saline.
Further, the initial seeding number of cells was 5×10 5 cells.
The beneficial effects of the invention are as follows:
The cell is continuously stimulated for a certain time by adopting the low-frequency sine wave with specific amplitude, so that the exosome yield is remarkably improved, the problems that the traditional exosome yield improvement method is low in efficiency, high in cost and difficult to meet production and living needs are solved, the cell is not damaged, the operation is convenient and fast, the cost is low, the automation degree is high, and the method is suitable for engineering application and popularization.
Drawings
FIG. 1 is a diagram showing the relationship between the concentration of exosomes and the amplitude of sound waves according to an embodiment of the present disclosure;
FIG. 2 is a schematic diagram of an exosome with 80 μm amplitude and a resting state observed under a transmission electron microscope according to an embodiment of the present disclosure;
FIG. 3 is a schematic diagram showing the particle size distribution of NTA particles according to one embodiment of the present disclosure; a is in a static state, b is in a vibration state;
FIG. 4 is a schematic diagram of Western blot protein semi-quantification in an embodiment of the present disclosure;
FIG. 5 is a schematic diagram showing the relative protein amounts of CD63 measured by Western blot in an embodiment of the present disclosure;
FIG. 6 is a schematic diagram showing the relative protein amounts of CD81 measured by Western blot in an embodiment of the present disclosure;
FIG. 7 is a schematic representation of cells under resting and vibrating conditions observed under transmission electron microscopy in an embodiment of the present disclosure; i is a static condition, and II is an acoustic vibration condition;
FIG. 8 is a graph showing MVB content observed under a transmission electron microscope in an embodiment of the present disclosure;
FIG. 9 is a graph showing ILV content observed under transmission electron microscopy in an embodiment of the present disclosure;
FIG. 10 is a graph showing the viability of A549 cells under different amplitude conditions in one embodiment of the present disclosure;
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
The following describes in detail the technical solutions provided by the embodiments of the present specification with reference to the accompanying drawings.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In one embodiment of the present disclosure, the apparatus is used to apply sound waves of a specific frequency and specific amplitude to cells in order to achieve a method of increasing the output of exosomes by acoustic wave vibration, and the apparatus includes a signal modulator, a power amplifier and a mediator.
An embodiment of the present disclosure provides a method for improving the output of exosomes by using acoustic wave vibration, the method specifically includes the following steps:
step one, carrying out subculture on the cells, placing the cells in a 37 ℃ incubator for 24 hours to wait for cell adhesion, discarding the original culture solution after cell adhesion, cleaning the cells, and culturing the cells by using the exosome-free fetal bovine serum.
It should be noted that, one implementation manner of the first step may be:
A549 cells were subcultured at an initial seeding density of 5×10 5/mL and placed in a 37 ℃ incubator for 24h waiting for cell attachment. After the cells had attached, the stock culture was discarded, washed twice with 2mL PBS, and a549 cells were cultured with 5mL exosome-free bovine serum.
Placing the cell culture bottle on a loudspeaker, playing sine waves with specific frequency and specific amplitude by the loudspeaker, culturing cells under the condition of sound wave vibration until the preset collection condition is reached, collecting cell supernatant and replacing a new exosome-free culture medium.
Wherein the frequency range of the low-frequency sine wave sound wave is 50 Hz-200 Hz, the amplitude range is 20-100 μm, and the application time is 6 h-72 h.
In order to achieve the optimal effect, a low-frequency sine wave acoustic wave can be applied by adopting the following system:
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 20 μm;
Sound wave 6 h with frequency of 100 Hz-200 Hz and amplitude of 40 μm;
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 60 μm;
Sound wave 12 h with frequency of 100 Hz-200 Hz and amplitude of 80 μm;
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 60 μm;
Sound wave 6 h with frequency of 100 Hz-200 Hz and amplitude of 40 μm;
sound wave 6 h with a frequency of 50 Hz-100 Hz and an amplitude of 20 μm.
The frequency in the system accords with the frequency of normal activities of a human body, the amplitude simulates the volume of speaking of the human body, the environment in the human body can be simulated, and the system has practical significance.
It should be noted that, one implementation manner of the second step may be:
The cell culture flask was placed on a speaker which played sound waves with a frequency of 150Hz and an amplitude of 20 μm, and the cells were cultured under the condition of sound wave vibration until a preset collection condition was reached, 5mL of cell supernatant was collected and replaced with a new exosome-free medium.
Another implementation manner of the second step may be:
the cell culture flask was placed on a speaker which played sound waves with a frequency of 150Hz and an amplitude of 40 μm, and the cells were cultured under the condition of sound wave vibration until a preset collection condition was reached, 5mL of cell supernatant was collected and replaced with a new exosome-free medium.
Yet another implementation manner of the second step may be:
The cell culture flask was placed on a speaker which played sound waves with a frequency of 150Hz and an amplitude of 60 μm, and the cells were cultured under the condition of sound wave vibration until a preset collection condition was reached, 5mL of cell supernatant was collected and replaced with a new exosome-free medium.
Yet another implementation manner of the second step may be:
the cell culture flask was placed on a speaker which played sound waves with a frequency of 150Hz and an amplitude of 80 μm, and the cells were cultured under the condition of sound wave vibration until a preset collection condition was reached, 5mL of cell supernatant was collected and replaced with a new exosome-free medium.
Yet another implementation manner of the second step may be:
The cell culture flask was placed on a speaker which played sound waves with a frequency of 150Hz and an amplitude of 100 μm, and the cells were cultured under the condition of sound wave vibration until a preset collection condition was reached, 5mL of cell supernatant was collected and replaced with a new exosome-free medium.
And thirdly, placing the cell supernatant collected in the second step into a centrifuge tube, performing centrifugal treatment for a plurality of times, sequentially removing cell fragments and dead cells, microbubbles or protein aggregates, filtering the cell supernatant by a filter, removing apoptotic bodies and microbubbles, separating exosomes from the supernatant by using an exosome separating instrument, and obtaining an exosome concentrated solution, and removing impurities in the exosome concentrated solution.
It should be noted that, one implementation manner of the third step may be:
5mL of the cell culture supernatants at 20, 40, 60, 80 and 100 μm amplitudes collected at each period were collected into 50mL centrifuge tubes, and centrifuged at 2000g at 4℃for 30min to remove cell debris and dead cells. The supernatant after centrifugation was centrifuged at 12000g for 30min to remove microbubbles or protein aggregates, and the supernatant obtained in the above step was filtered with a 0.22 μm filter to remove apoptotic bodies and microbubbles, and collected in a 15mL centrifuge tube. The exosomes were then separated from the supernatant using EXODUS (exosome separation instrument), and the concentrate was washed twice with 1mL of PBS to further remove impurities. Finally, the isolated exosomes were resuspended in 200 μl of PBS rinse and prepared for further analysis.
The following tests were all performed for step two using the exosomes obtained under the following regimen conditions, unless otherwise specified:
The following system is adopted:
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 20 μm;
Sound wave 6 h with frequency of 100 Hz-200 Hz and amplitude of 40 μm;
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 60 μm;
Sound wave 12 h with frequency of 100 Hz-200 Hz and amplitude of 80 μm;
Sound wave 6 h with frequency of 50 Hz-100 Hz and amplitude of 60 μm;
Sound wave 6 h with frequency of 100 Hz-200 Hz and amplitude of 40 μm;
sound wave 6 h with a frequency of 50 Hz-100 Hz and an amplitude of 20 μm.
And (3) primarily quantifying the related total protein of the exosome under all amplitudes by using a BCA kit, and quantifying the early morphology of the exosome by using a transmission electron microscope after finding out the optimal condition.
It should be noted that the following procedure may also be adopted:
The collected exosomes associated total proteins were initially quantified using BCA kit to explore optimal conditions. As a result, as shown in FIG. 1, the exosome production was higher than that of the stationary culture under the condition of applying vibration of different amplitudes. Wherein the yield is improved most under the stimulation of low-frequency sine wave sound wave with the amplitude of 80 mu m, so that the stimulation of 80 mu m is used as the optimal condition.
Transmission electron microscopy was used to image early forms of intracellular exosome production. We used transmission electron microscopy to image the internal cell structures. We observed typical multivesicular bodies (MVBs) and endoluminal vesicles (ILVs) within the cells and found that the number of MVBs under shaking culture was much higher than that under static culture (fig. 7). In resting state (fig. 8), the average number of MVBs per cell was 1.2±0.2; the number of vibration states (fig. 9) was 4.8±1.1, which is about 4 times that of the rest state. While total ILV number per cell was 28.0.+ -. 8.1 at rest and vibration was 79.4.+ -. 17.1 at rest, approximately 3 times.
The exosomes were characterized by transmission electron microscopy, NTA, WB, and the improvement in exosome production was again demonstrated by WB semi-quantitative results.
For the characteristics of exosomes generated in a static state and under the condition of adopting acoustic wave vibration, TEM (transmission electron microscope) observation of morphological characteristics, NTA (non-linear alumina) observation of particle size and potential and Western Blot (WB) observation of characteristic proteins are carried out. As shown in fig. 2, the exosomes observed under the transmission electron microscope in the static state and the vibration state are both cup-stand-shaped, and conform to the normal exosome shape. As shown in fig. 3, the particle size distribution was drawn from the NTA particle size measurement results, the particle size distribution was within 200nm in both static and vibrating states, and the average particle size of exosomes was around 150nm under all conditions, without significant difference. As shown in fig. 4, WB results showed that calnexin was present in cells as an endoplasmic reticulum marker only, and that no expression was present in exosomes, indicating that exosomes were not contaminated with cell debris during purification. The expression of both CD63 and CD81 proteins under vibration was higher than that at rest and up to about 3-fold (fig. 5, 6), demonstrating that the method increased exosome production by 3-4 fold.
The cell viability after applying the sound to the cells was measured as a result of the stimulation with sound waves having a frequency of 150Hz and an amplitude of 20 μm, 40 μm, 60 μm, 80 μm, 100 μm, respectively.
As shown in FIG. 10, after 72 hours of cell culture, viability of cells under static and varying vibration amplitude acoustic waves was measured using live/dead staining, and from the results, it can be seen that the cell viability remained above 90% after the acoustic wave stimulation was applied to the cells, indicating that the acoustic wave vibration did not adversely affect the cell viability, and was harmless. The survival rate of the vibrating cells is more than 95% by adopting the system, and the result is unattached.
In summary, the invention obviously improves the exosome yield by adopting the low-frequency sine wave with specific amplitude to continuously stimulate the cells for a certain time, wherein after the acoustic wave stimulation is applied by adopting the specific system of the invention, the exosome yield is improved by 3 times to 4 times compared with the resting secretion, and in the same time, the exosome yield of the method is far higher than that of the exosome yield of the traditional exosome yield improving method, and the problems that the traditional exosome yield improving method is low in efficiency, high in cost and difficult to meet the production and living needs are solved; through verification, the exosomes produced by the method have the structure and the function of normal exosomes, do not harm cells, can be continuously produced, have higher safety and higher stability, further reduce the complexity of exosome production procedures while ensuring that the yield of the exosomes meets the production requirement, and have important social and economic significance and clinical value; the method is convenient to operate, low in cost, high in automation degree and suitable for engineering application and popularization.
The present invention is not limited to the above-mentioned embodiments, but is intended to be limited to the following embodiments, and any modifications, equivalents and modifications can be made to the above-mentioned embodiments without departing from the scope of the invention.
Claims (9)
1. A method for improving the output of exosomes by using acoustic wave vibration, which is characterized by comprising the following steps: subculturing the cells; judging whether cells adhere to the wall, if so, discarding the original culture solution; culturing cells with exosome-free foetal calf serum after washing; applying a preset low-frequency sine wave sound wave to the process of culturing cells by adopting exosome-free fetal bovine serum to obtain a cell culture solution; collecting cell culture solution, and centrifuging to obtain exosomes; the frequency range of the low-frequency sine wave sound wave is 50 Hz-200 Hz, the amplitude range is 20 mu m-100 mu m, and the application time is 6h-72h.
2. The method for improving the output of exosomes by using acoustic wave vibration according to claim 1, wherein the process of applying low-frequency sine wave acoustic wave is sequentially carried out according to the following system:
sound wave with frequency of 50 Hz-100 Hz and amplitude of 20 μm for 6h;
sound wave with frequency of 100 Hz-200 Hz and amplitude of 40 μm for 6h;
Sound wave with frequency of 50 Hz-100 Hz and amplitude of 60 μm for 6h;
Sound wave with frequency of 100 Hz-200 Hz and amplitude of 80 μm for 12h;
Sound wave with frequency of 50 Hz-100 Hz and amplitude of 60 μm for 6h;
sound wave with frequency of 100 Hz-200 Hz and amplitude of 40 μm for 6h; the frequency is 50 Hz-100 Hz,
The sound wave with the amplitude of 20 μm is 6h.
3. The method for increasing the output of exosomes by using acoustic wave vibration according to any one of claims 1 or 2, wherein the mode of applying low frequency sine wave acoustic wave is as follows:
Placing a speaker under the cell culture vessel;
controlling the continuous playing frequency and amplitude of the loudspeaker to meet the set requirements;
Judging whether the playing time of the loudspeaker reaches a preset time threshold, and if so, collecting the cell culture fluid.
4. The method for increasing the output of an exosome by acoustic vibration according to claim 2 wherein the frequency, amplitude and time of the speaker play are controlled by a mobile digital terminal.
5. The method for improving the output of exosomes by using acoustic wave vibration according to claim 1, wherein the centrifugation process is as follows:
centrifuging the cell culture solution by using a relative centrifugal force of 2000g to obtain a supernatant from which cell fragments and dead cells are removed;
centrifuging the supernatant from which cell debris and dead cells are removed by using a relative centrifugal force of 12000g to obtain a supernatant from which microbubbles or protein aggregates are removed;
Filtering the supernatant from which the microbubbles or protein aggregates are removed by using a 0.22 μm filter to obtain a supernatant from which apoptotic bodies and microbubbles are removed;
and adding the supernatant with the apoptotic bodies and microbubbles removed into an exosome separation instrument for separation to obtain exosomes.
6. The method of claim 5, further comprising the steps of, after obtaining the exosomes: the exosomes were washed with phosphate buffered saline and then resuspended with phosphate buffered saline.
7. The method for increasing exosome production by sonic vibration according to claim 1, wherein the cell subculture conditions are as follows: cells were placed in an incubator at 37℃and subcultured for 24 hours.
8. The method of claim 1, wherein the step of washing the cells comprises the steps of: cells were washed twice with phosphate buffered saline.
9. The method of claim 1, wherein the initial cell seeding number is 5 x 10 5 cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310479553.9A CN116716288B (en) | 2023-04-28 | 2023-04-28 | Method for improving exosome yield by acoustic wave vibration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310479553.9A CN116716288B (en) | 2023-04-28 | 2023-04-28 | Method for improving exosome yield by acoustic wave vibration |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116716288A CN116716288A (en) | 2023-09-08 |
| CN116716288B true CN116716288B (en) | 2024-04-26 |
Family
ID=87872253
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310479553.9A Active CN116716288B (en) | 2023-04-28 | 2023-04-28 | Method for improving exosome yield by acoustic wave vibration |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116716288B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676065A (en) * | 2017-03-10 | 2017-05-17 | 四川大学 | Adipose tissue source exosome gel, preparation method and application |
| WO2017155166A1 (en) * | 2016-03-11 | 2017-09-14 | 가톨릭관동대학교기술지주 주식회사 | Cell reprogramming method using imposition of physical stimulation-mediated environmental transition |
| CN110777142A (en) * | 2019-06-27 | 2020-02-11 | 上海交通大学 | Method for promoting secretion of extracellular fluid by low-intensity pulse ultrasonic stimulation |
| CN111363679A (en) * | 2018-12-26 | 2020-07-03 | 浙江大学 | Method for stimulating large-scale secretion of exosomes by cells |
| CN111363680A (en) * | 2018-12-26 | 2020-07-03 | 浙江大学 | Pulse stirring type bioreactor for secretion, separation and collection of exosome |
| WO2021114164A1 (en) * | 2019-12-12 | 2021-06-17 | 中国科学院深圳先进技术研究院 | Exosome, preparation method therefor and use thereof |
| CN113061579A (en) * | 2019-12-12 | 2021-07-02 | 中国科学院深圳先进技术研究院 | Exosome and preparation method and application thereof |
| CN113684130A (en) * | 2021-08-17 | 2021-11-23 | 深圳高性能医疗器械国家研究院有限公司 | Equipment and method for stimulating secretion of cell exosome, obtained exosome and application thereof |
| CN114107205A (en) * | 2021-11-29 | 2022-03-01 | 厦门大学附属翔安医院 | Method for stimulating cells to quickly secrete exosomes and application thereof |
| JP2022121333A (en) * | 2021-02-08 | 2022-08-19 | Ｕｂｅ株式会社 | Exosome production method |
| CN114958721A (en) * | 2022-06-16 | 2022-08-30 | 刘萍 | Extraction method of stem cell exosomes |
| WO2023019431A1 (en) * | 2021-08-17 | 2023-02-23 | 深圳高性能医疗器械国家研究院有限公司 | Device and method for stimulating secretion of cellular exosomes, and obtained exosomes and application thereof |
-
2023
- 2023-04-28 CN CN202310479553.9A patent/CN116716288B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017155166A1 (en) * | 2016-03-11 | 2017-09-14 | 가톨릭관동대학교기술지주 주식회사 | Cell reprogramming method using imposition of physical stimulation-mediated environmental transition |
| CN106676065A (en) * | 2017-03-10 | 2017-05-17 | 四川大学 | Adipose tissue source exosome gel, preparation method and application |
| CN111363679A (en) * | 2018-12-26 | 2020-07-03 | 浙江大学 | Method for stimulating large-scale secretion of exosomes by cells |
| CN111363680A (en) * | 2018-12-26 | 2020-07-03 | 浙江大学 | Pulse stirring type bioreactor for secretion, separation and collection of exosome |
| CN110777142A (en) * | 2019-06-27 | 2020-02-11 | 上海交通大学 | Method for promoting secretion of extracellular fluid by low-intensity pulse ultrasonic stimulation |
| WO2021114164A1 (en) * | 2019-12-12 | 2021-06-17 | 中国科学院深圳先进技术研究院 | Exosome, preparation method therefor and use thereof |
| CN113061579A (en) * | 2019-12-12 | 2021-07-02 | 中国科学院深圳先进技术研究院 | Exosome and preparation method and application thereof |
| JP2022121333A (en) * | 2021-02-08 | 2022-08-19 | Ｕｂｅ株式会社 | Exosome production method |
| CN113684130A (en) * | 2021-08-17 | 2021-11-23 | 深圳高性能医疗器械国家研究院有限公司 | Equipment and method for stimulating secretion of cell exosome, obtained exosome and application thereof |
| WO2023019431A1 (en) * | 2021-08-17 | 2023-02-23 | 深圳高性能医疗器械国家研究院有限公司 | Device and method for stimulating secretion of cellular exosomes, and obtained exosomes and application thereof |
| CN114107205A (en) * | 2021-11-29 | 2022-03-01 | 厦门大学附属翔安医院 | Method for stimulating cells to quickly secrete exosomes and application thereof |
| CN114958721A (en) * | 2022-06-16 | 2022-08-30 | 刘萍 | Extraction method of stem cell exosomes |
Non-Patent Citations (2)
| Title |
|---|
| Regulation of exosomes secretion by low-intensity pulsed ultrasound in lung cancer cells;Qingyu Zeng等;Experimental Cell Research;20191001;第383卷(第1期);1-8 * |
| 动物外泌体分离方法的研究进展;邱金戈;刘德武;孙宝丽;李耀坤;郭勇庆;邓铭;柳广斌;;中国生物工程杂志;20200915(09);41-47 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116716288A (en) | 2023-09-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11730768B2 (en) | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids | |
| CN114591905B (en) | Method for preparing apoptotic vesicles from human erythrocytes and application of apoptotic vesicles | |
| WO2020103192A1 (en) | Method for extracting exosome | |
| CN107007541A (en) | Application of the excretion body in skin-whitening preparation | |
| CN107988153A (en) | The method of mesenchymal stem cells derived from human umbilical blood source separation excretion body and the reagent used | |
| CN111467373A (en) | Dental pulp stem cell exosome preparation, preparation method and application thereof | |
| CN112501112A (en) | Separation and enrichment method for rapidly extracting tissue extracellular vesicles | |
| CN116716288B (en) | Method for improving exosome yield by acoustic wave vibration | |
| CN111117965A (en) | Rapid purification method of high-purity primary tumor cells | |
| CN113388570B (en) | Bovine rumen epithelial tissue dissociation method for single cell sequencing | |
| CN108486039B (en) | Method for inducing human adipose-derived stem cells to differentiate into testicular interstitial cells by using small molecules | |
| WO2023019431A1 (en) | Device and method for stimulating secretion of cellular exosomes, and obtained exosomes and application thereof | |
| CN113583952A (en) | Culture solution for increasing yield of exosomes of stem cells | |
| Ferroni et al. | Methods to isolate adipose tissue-derived stem cells | |
| CN112852725A (en) | Preparation method and application for extracting and purifying stem cell exosome by using protein cross-linked nano affinity microspheres | |
| CN114703120B (en) | Separation method of animal nervous system vascular smooth muscle cell single cells | |
| Romanov et al. | Human umbilical cord mesenchymal stromal cell-derived microvesicles express surface markers identical to the phenotype of parental cells | |
| CN111321117A (en) | Method for three-dimensional culture and expansion of hematopoietic stem cells | |
| CN115537389A (en) | Preparation method of umbilical cord mesenchymal stem cell source exosome with inflammation regulation function | |
| CN115558638A (en) | Exosome prepared from placenta mesenchymal stem cells and application thereof | |
| CN114657184A (en) | Multivalent aptamer functionalized DNA nano-structure probe and preparation method and application thereof | |
| CN111876372A (en) | One-step separation method of mouse liver parenchymal cells and non-parenchymal cells | |
| CN114085811B (en) | Method for in-vitro expansion of endometrial mesenchymal stem cells by utilizing myometrium cell exosomes | |
| WO2021114160A1 (en) | Method for producing microvesicles, and microvesicles obtained based on the method for producing microvesicles and application thereof | |
| CN112662618B (en) | Umbilical cord mesenchymal stem cells and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |