CN113249302A - Efficient exosome separation and purification method - Google Patents
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Abstract
The invention relates to the technical field of molecular biology, in particular to a high-efficiency exosome separation and purification method, which comprises the steps of separating plasma, adding a cationic polymer for incubation; the filter membrane entraps the exosomes bound to the cationic polymer and rinses the exosomes; eluting exosomes with chaotropic agent; adding magnetic beads for adsorption and enriching exosomes; eluting to obtain an exosome concentrated solution. Compared with the prior art, the invention does not use large-scale centrifugal equipment, adopts the cationic polymer to coat the exosome, adopts the chaotropic agent to relieve the action of the cationic polymer and the exosome, realizes exosome recovery through simple filtration, and then concentrates and purifies the recovered exosome through the magnetic adsorbent. The method effectively shortens the separation time, improves the recovery efficiency of the exosomes, improves the flux of the processed samples, improves the separation purity of the exosomes, reduces the cost of reagents and consumables required by the exosome separation, and the high-purity exosomes obtained by separation can be used for experiments such as nucleic acid extraction Westernblot, particle size analysis, electron microscope detection and the like.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a high-efficiency exosome separation and purification method.
Background
Exosomes (Exosomes) are extracellular vesicles of a circular monolayer membrane structure with the diameter of 30-150 nm, which are formed by cells through a series of regulation processes of endocytosis, fusion, efflux and the like. Exosomes are released by numerous types of cells in the body, are widely distributed in body fluids such as saliva, plasma, milk, urine, and the like, can carry proteins, transport RNA, and play an important role in intercellular substance and information transduction. The discovery of exosomes enriches the understanding of researchers on the communication mode between cells, deepens the understanding of the physiological and pathological processes of organisms, and three scientists which have outstanding contribution to cell vesicles are awarded in the 2013 Nobel biology/medicine prize, so that the research on exosomes reaches the brand-new height.
In recent years, as the research and the recognition of exosomes are deepened, exosome detection is widely applied to noninvasive diagnosis, treatment and monitoring of tumors and diseases by a plurality of clinical scientific research institutions as a novel liquid biopsy hotspot technology; how to extract exosomes efficiently is the key to realizing the clinical routine application of the emerging liquid biopsy technology. Exosomes are one of three fields of liquid biopsy, and potential biological targets of exosomes are numerous, but trace nucleic acid and protein can be simultaneously applied to exosome separation technology, generally, in order to obtain high-purity exosomes.
The prior separation technology of exosome comprises ultracentrifugation, molecular exclusion, immunocapture, PEG precipitation and other methods. Ultracentrifugation generally needs more than 7 hours, and is time-consuming and labor-consuming, and required instruments and equipment are expensive, so that a sufficient amount of exosomes are difficult to obtain for subsequent experimental analysis; the size exclusion method, namely the immunocapture method, has little amount of collected exosomes and high cost; although the PEG precipitation method can separate the exosome on a large scale, the lipoprotein is inevitably introduced while a large amount of exosome is collected, so that the problem of low purity of the exosome obtained by separation is caused. Because of the shortage of exosome extraction reagents in China, the research on exosomes in China basically depends on the ultracentrifugation and import extraction kit with complicated processes, so the development of the research and clinical application of exosomes in China is seriously hindered. How to seek a mode capable of efficiently separating exosomes and ensuring the purity of exosomes is an urgent need in the technical field.
Disclosure of Invention
In view of the above-mentioned drawbacks of the background art, the present invention provides a method for separating and purifying exosomes with high efficiency.
The technical scheme adopted by the invention is as follows: a high-efficiency exosome separation and purification method is characterized by comprising the following steps:
s1, adding the cationic polymer into the body fluid, and incubating for 5-30 min at 2-10 ℃;
s2, filtering the body fluid mixture, and adding a low-concentration chaotropic agent and a high-concentration chaotropic agent into the retentate in sequence for elution to obtain an exosome primary isolate;
s3, adding a magnetic adsorbent into the primary exosome isolate, incubating at 15-35 ℃ for 2-20 min, removing clear liquid, and taking out the magnetic adsorbent;
and S4, adding ammonia water into the magnetic adsorbent for resuspension, incubating at 15-35 ℃ for 2-20 min, and taking out clear liquid to obtain the high-purity exosome.
Preferably, the physiological fluid comprises the following fluids: nasopharynx, oral cavity, esophagus, stomach, pancreas, liver, pleura, pericardium, peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph, plasma, serum, synovial fluid, cerebrospinal fluid, uterine cavities and appendages, urine, and interstitial, intracellular and extracellular fluids; blood, plasma and serum are preferred.
Preferably, the cationic polymer in S1 is polylysine.
Preferably, S2 is filtered through a filter membrane with a pore size of 0.1-0.3 μm.
Preferably, the low concentration chaotropic agent in S2 is 0.5-2.5M guanidinium isothiocyanate.
Preferably, the chaotropic agent at a high concentration in S2 is 4-6M guanidinium isothiocyanate.
Preferably, the magnetic adsorbent in S3 is a magnetic bead coated with titanium dioxide.
Preferably, the ammonia water in S4 is 5-25% ammonia water.
Preferably, the magnetic adsorbent in S4 is rinsed with PBS before resuspension.
Has the advantages that: compared with the prior art, the high-efficiency exosome separation and purification method provided by the invention has the advantages that large-scale centrifugal equipment is not used, the exosome is coated by adopting the cationic polymer, the exosome is rinsed by adopting the low-concentration chaotropic agent, the action of the cationic polymer and the exosome is relieved by adopting the high-concentration chaotropic agent, the exosome recovery is realized by simple filtration, and then the recovered exosome is concentrated and purified by the magnetic adsorbent. The method effectively shortens the separation time, improves the recovery efficiency of the exosomes, improves the flux of the processed samples, improves the separation purity of the exosomes, and reduces the cost of reagents and consumables required by the exosome separation.
Drawings
FIG. 1 is a graph comparing the total protein concentration after purification of exosomes obtained from each exosome separation method;
FIG. 2 is a graph comparing the recovery efficiency of each exosome separation method;
FIG. 3 is a comparison graph of the total amount of protein obtained by each exosome separation method after purification;
FIG. 4 is a graph comparing the exosome purities obtained for each exosome separation method;
FIG. 5 is a graph comparing the separation duration for each exosome separation method.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Example 1 a high efficiency method for exosome isolation and purification comprising the steps of:
s1, adding the cationic polymer into the plasma, and incubating for 5min at 2 ℃;
s2, extracting the plasma mixture by using a syringe, connecting a filter membrane with the aperture of 0.1, pushing the syringe for filtration, intercepting exosome combined with the cationic polymer by using the filter membrane, disconnecting the syringe, absorbing 0.5M guanidinium isothiocyanate, connecting the filter membrane with the aperture of 0.1, pushing the syringe for filtration, disconnecting the syringe, absorbing 4M guanidinium isothiocyanate, connecting the filter membrane with the aperture of 0.1, pushing the syringe for filtration, and collecting filtrate to obtain primary exosome isolate;
s3, adding magnetic beads wrapped with titanium dioxide into the primary exosome isolate, performing shaking incubation at 15 ℃ and 1000rpm for 2min, placing on a magnetic frame, settling the magnetic beads for 1min, and removing supernatant;
s4, rinsing the magnetic beads with PBS, adding 5% ammonia water for resuspension, carrying out shaking incubation at 15 ℃ and 1000rpm for 5min, placing on a magnetic frame, settling the magnetic beads for 1min, and transferring the supernatant to a new tube to obtain the high-purity exosome.
Example 2 a high efficiency method for exosome isolation and purification, comprising the following steps:
s1, adding the cationic polymer into the plasma, and incubating for 30 min at 10 ℃;
s2, extracting the plasma mixture by using a syringe, connecting a filter membrane with the aperture of 0.3, pushing the syringe for filtration, intercepting exosome combined with the cationic polymer by using the filter membrane, disconnecting the syringe, absorbing 2.5M guanidinium isothiocyanate, connecting the filter membrane with the aperture of 0.3, pushing the syringe for filtration, disconnecting the syringe, absorbing 4M guanidinium isothiocyanate, connecting the filter membrane with the aperture of 0.3, pushing the syringe for filtration, and collecting filtrate to obtain primary exosome isolate;
s3, adding magnetic beads wrapped with titanium dioxide into the primary exosome isolate, performing shaking incubation at 35 ℃ and 1000rpm for 20min, placing on a magnetic frame, settling the magnetic beads for 1min, and removing supernatant;
s4, rinsing the magnetic beads with PBS, adding 5% ammonia water for resuspension, oscillating and incubating at 35 ℃ and 1000rpm for 20min, placing on a magnetic frame, settling the magnetic beads for 1min, and transferring the supernatant to a new tube to obtain the high-purity exosome.
Example 3 a high efficiency method for exosome isolation and purification, comprising the following steps:
s1, adding the cationic polymer into the plasma, and incubating for 20min at 4 ℃;
s2, extracting the plasma mixture by using a syringe, connecting a filter membrane with the aperture of 0.2, pushing the syringe for filtration, intercepting exosome combined with the cationic polymer by the filter membrane, disconnecting the syringe, absorbing 2M guanidinium isothiocyanate, connecting the filter membrane with the aperture of 0.2, pushing the syringe for filtration, disconnecting the syringe, absorbing 5M guanidinium isothiocyanate, connecting the filter membrane with the aperture of 0.2, pushing the syringe for filtration, and collecting filtrate to obtain an exosome primary isolate;
s3, adding magnetic beads wrapped with titanium dioxide into the primary exosome isolate, performing shaking incubation at 25 ℃ and 1000rpm for 10min, placing on a magnetic frame, settling the magnetic beads for 1min, and removing supernatant;
s4, rinsing the magnetic beads with PBS, adding 5% ammonia water for resuspension, carrying out shaking incubation at 25 ℃ and 1000rpm for 10min, placing on a magnetic frame, settling the magnetic beads for 1min, and transferring the supernatant to a new tube to obtain the high-purity exosome.
Comparative example 1 separation by UC method
Centrifuging plasma 300 g at 4 deg.C for 10min, and transferring supernatant to new tube; centrifuging at 4 deg.C for 20min at 10000 g, and filtering the supernatant with 0.22 μm filter membrane; 100000 g, 4 deg.C, 90 min, removing supernatant; 1mL of cold PBS resuspended pellet, 100000 g again, 4 ℃, 90 min, 100 μ L of cold PBS resuspended pellet to obtain exosomes.
Comparative example 2 separation by FC
Centrifuging plasma 300 g at 4 deg.C for 10min, and transferring supernatant to new tube; centrifuging at 4 deg.C for 20min at 10000 g, and filtering the supernatant with 0.22 μm filter membrane; and adding the filtered liquid into an Amicon Ultra 100KD ultrafiltration tube, adding 4mL of PBS, uniformly mixing, 3000 g, centrifuging until the residual 200 mu L of liquid is stopped, adding 4mL of PBS again, centrifuging until the residual 200 mu L of liquid is stopped, and transferring the liquid to a new tube to obtain the separated exosome.
Comparative example 3 separation by IP method
Taking 300 g of pulp, centrifuging at 4 ℃ for 10min, and transferring supernatant to a new tube; centrifuging at 4 deg.C for 20min at 10000 g, and filtering the supernatant with 0.22 μm filter membrane; adding 10 μ L of CD9/CD63/CD81 magnetic beads which are coupled with PBS in a balanced manner, shaking and mixing uniformly at 4 ℃ for 60 min, placing the centrifugal tube on a magnetic frame for 1min, and removing supernatant; taking down the centrifugal tube from the magnetic frame, adding 1mL of PBS (phosphate buffer solution) for resuspending the magnetic beads, placing the centrifugal tube on the magnetic frame again for 1min, and removing the supernatant; taking down the centrifugal tube from the magnetic frame, adding 100 μ L of 0.1M Glysineph3.0, blowing, beating, mixing, and incubating at room temperature for 5 min; placing the centrifuge tube on a magnetic frame for 1min, transferring the supernatant to a new tube, and adding 400 mu LPBS to obtain the exosome.
Comparative example 4 separation by PEG method
Centrifuging 200 μ L plasma 300 g at 4 deg.C for 10min, and transferring supernatant to new tube; centrifuging at 4 deg.C for 20min at 10000 g, and filtering the supernatant with 0.22 μm filter membrane; add 100. mu.L LPBS and 60. mu.L precipitant (invitrogen) to the solution and incubate for 30 min at 4 ℃; 10000 g, 5min centrifugation, removing supernatant; to the pellet was added 100 μ L PBS and pipetted to resuspend the exosomes.
The recovery rate, total protein concentration and content of exosomes isolated using the methods of example 3 and comparative examples 1-4 were determined.
Protein concentration determination: the protein concentration of the exosome solution after separation was determined by Bradford method after the exosome solution was uniformly diluted to 500 μ L. In FIG. 1, it is shown that the total protein concentration of the exosomes isolated by the present invention is higher than that of comparative examples 1-3 and lower than that of the PEG method employed in comparative example 4.
And (3) testing the recovery rate: the protein concentration of the separated exosome solution was measured by the Bradford method, and a total amount of 50. mu.g of protein solution was taken according to the measured concentration, subjected to polyacrylamide gel electrophoresis, and detected by hybridization with CD9/CD63/TSG101 antibodies, respectively. In FIG. 2 it is shown that the present invention has much higher exosome recovery than comparative examples 1-3, and slightly higher recovery than the PEG method used in comparative example 4.
And (3) measuring total exosome amount: and (4) measuring the particle content in the solution by using a zetaview particle size detector. In fig. 3 it is shown that the total amount of exosomes isolated by the present invention is higher than in comparative examples 1-4.
And (3) measuring the purity of the exosome: the number of vesicles was divided by the total protein content and normalized using the UC ratio as 1 to obtain the separation purity ratio. In fig. 4 it is shown that the exosomes isolated by the present invention are of higher purity than comparative examples 1, 2, 4, slightly lower than the IP process used in comparative example 3.
FIG. 5 shows that the separation of exosomes by the method of the present invention takes less than 40 min in the whole process, which is shorter than the separation time of comparative examples 1-4, and the operation is convenient.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Claims (9)
1. A high-efficiency exosome separation and purification method is characterized by comprising the following steps:
s1, adding the cationic polymer into the body fluid, and incubating for 5-30 min at 2-10 ℃;
s2, filtering the body fluid mixture, and adding a low-concentration chaotropic agent and a high-concentration chaotropic agent into the retentate in sequence for elution to obtain an exosome primary isolate;
s3, adding a magnetic adsorbent into the primary exosome isolate, incubating at 15-35 ℃ for 2-20 min, removing clear liquid, and taking out the magnetic adsorbent;
and S4, adding an eluant into the magnetic adsorbent for heavy suspension, incubating at 15-35 ℃ for 2-20 min, and taking out clear liquid to obtain the high-purity exosome.
2. A high efficiency exosome separation and purification method according to claim 1, characterized by: the body fluid comprises blood, plasma or serum of a non-human neoplasm.
3. A high efficiency exosome separation and purification method according to claim 1, characterized by:
the cationic polymer in S1 is polylysine.
4. A high efficiency exosome separation and purification method according to claim 3, characterized by: s2 is filtered by a filter membrane with the pore diameter of 0.1-0.3 μm.
5. A high efficiency exosome separation and purification method according to claim 1, characterized by: the low concentration chaotropic agent in S2 is 0.5-2.5M guanidinium isothiocyanate.
6. A high efficiency exosome separation and purification method according to claim 1, characterized by: the high concentration chaotropic agent in S2 was 4-6M guanidinium isothiocyanate.
7. A high efficiency exosome separation and purification method according to claim 1, characterized by: the magnetic adsorbent in S3 is a magnetic bead wrapped with titanium dioxide.
8. A high efficiency exosome separation and purification method according to claim 1, characterized by: the eluent in S4 is 5-25% ammonia water.
9. A high efficiency exosome separation and purification method according to claim 1, characterized by: the magnetic adsorbent in S4 was rinsed with PBS before resuspension.
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Cited By (3)
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| CN114457004A (en) * | 2021-12-23 | 2022-05-10 | 江苏为真生物医药技术股份有限公司 | Exosome separation method in biological sample, kit and application thereof |
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| CN114702621A (en) * | 2022-04-12 | 2022-07-05 | 大连医科大学 | pH response random copolymer, preparation method thereof and exosome homogeneous phase separation and purification method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114457004A (en) * | 2021-12-23 | 2022-05-10 | 江苏为真生物医药技术股份有限公司 | Exosome separation method in biological sample, kit and application thereof |
| CN114457004B (en) * | 2021-12-23 | 2024-04-16 | 江苏为真生物医药技术股份有限公司 | Method for separating exosomes in biological sample, kit and application thereof |
| CN114574437A (en) * | 2022-01-19 | 2022-06-03 | 大连博格林生物科技有限公司 | Plasma exosome extraction reagent, enrichment method, extraction kit and application thereof |
| CN114702621A (en) * | 2022-04-12 | 2022-07-05 | 大连医科大学 | pH response random copolymer, preparation method thereof and exosome homogeneous phase separation and purification method |
| CN114702621B (en) * | 2022-04-12 | 2024-01-30 | 大连医科大学 | PH response random copolymer, preparation method thereof and exosome homogeneous phase separation and purification method |
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