CN113846048A - Method for extracting exosome - Google Patents
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Abstract
The invention is suitable for the technical field of extraction processes, and provides an extraction method of exosomes, which comprises the following steps: 1) preparation of PEG extract: weighing polyethylene glycol and sodium chloride, and fully dissolving in pure water to obtain an extracting solution; 2) cell culture: when the cell fusion degree is about 60% -70%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer, adding a fresh culture medium containing serum-free related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell fragments to obtain initial supernatant; 3) exosome extraction: centrifuging the initial supernatant in the step 2) twice, taking the supernatant, adding the extracting solution in the step 1), inverting, fully mixing, incubating, centrifuging, removing the supernatant, collecting the precipitate, suspending the precipitate by using a phosphate buffer solution, and storing a sample subgroup at-80 ℃. The invention has simple steps and low cost and can be produced in mass.
Description
Technical Field
The invention belongs to the technical field of extraction processes, and particularly relates to an exosome extraction method.
Background
Exosomes (exosomes) are one type of Extracellular Vesicles (EVs) secreted by living cells, have a diameter of about 30-200nm, a density of 1.13-1.21g/ml, have a typical lipid bilayer structure, and are released extracellularly after intracellular multivesicular bodies (MVBs) are fused with cell membranes. Almost all types of cells (immune cells, nerve cells, stem cells), including tumor cells, can produce and release exosomes, but exosomes from different cell sources contain different RNA, lipid, and protein components. Functional proteins, mRNA, microRNA and other substances contained in the exosome play an important role in the intercellular information transfer process, participate in the intercellular substance transportation and information transfer, and regulate and control the physiological activities of cells. Therefore, the exosome plays a great role in convenience of antigen presentation, immune escape, normal cell transformation induction, tumor occurrence and metastasis promotion and the like, and in addition, the exosome plays a key role in physiological processes such as immune response, inflammatory reaction, angiogenesis, apoptosis, blood coagulation, waste treatment and the like, and can be used as a natural nanoparticle for drug delivery.
At present, ultracentrifugation, filtration centrifugation and immunomagnetic bead methods are commonly used in exosome separation methods. However, these methods have certain disadvantages.
The ultracentrifugation operation is complicated, long in time and needs to consume a large amount of raw materials, the repeated centrifugation operation is likely to damage vesicles of exosomes so as to reduce the quality of the exosomes, or soluble proteins in a sample and the exosomes can form aggregates and lumps together so as to cause pollution.
The main disadvantages of filtration centrifugation are that exosomes may block the filtration pores, resulting in a reduced membrane life, a lower separation efficiency, and the exosome membranes may stick to each other, resulting in a low separation yield and even in erroneous detection results.
The magnetic bead immunization method has low efficiency, the bioactivity of the substances contained in the exosome is easily influenced by pH and salt concentration, the downstream experiment is not facilitated, the yield is extremely low, and the magnetic bead immunization method cannot be used commercially.
Disclosure of Invention
The embodiment of the invention aims to provide an exosome extraction method, and aims to solve the problem that existing ultracentrifugation, filtering centrifugation and immunomagnetic bead methods adopted in the prior art are insufficient in exosome extraction.
The embodiment of the invention is realized in such a way that the method for extracting the exosome comprises the following steps:
1) preparation of PEG extract: weighing polyethylene glycol and sodium chloride, and fully dissolving in pure water to obtain an extracting solution;
2) cell culture: when the cell fusion degree is about 60% -70%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer, adding a fresh culture medium containing serum-free related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell fragments to obtain initial supernatant;
3) exosome extraction: centrifuging the initial supernatant in the step 2) twice, taking the supernatant, adding the extracting solution in the step 1), inverting, fully mixing, incubating at 4-37 ℃ for 10-20 hours, centrifuging for the third time, discarding the supernatant, collecting the precipitate, suspending the precipitate with phosphate buffer, and storing the sample subset at-80 ℃.
The embodiment of the invention provides an exosome extraction method, which comprises the following steps:
1. the steps are few and simple, and the exosome concentrated solution can be collected only by twice centrifugation, once incubation and once centrifugation;
2. the cost is low, and only polyethylene glycol, sodium chloride and a high-speed centrifuge (10000g) are needed. Ultra-high speed centrifuges (200000g), porous filters, or immunomagnetic beads are not required.
3. Can be produced in mass production, does not need complex equipment and expensive preparations, can process a large amount of samples each time and obtain a large amount of exosomes.
Drawings
FIG. 1 is a scanning electron microscope image of exosomes extracted in the example of the present invention;
fig. 2 is a diagram illustrating a particle size distribution of exosomes extracted in the example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
Example 1
An extraction method of exosomes, comprising the steps of:
1) preparation of PEG extract: weighing polyethylene glycol with the molecular weight of 4000 and the mass of 10g and sodium chloride with the mass of 29.25g, and fully dissolving the polyethylene glycol and the sodium chloride in pure water to obtain an extracting solution, wherein the concentration of the polyethylene glycol in the extracting solution is 0.1g/L, and the concentration of the sodium chloride is 0.5 mol/L;
2) cell culture: when the cell fusion degree is about 60%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer solution with the pH value of 7.4, adding a fresh culture medium without serum related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell debris to obtain initial supernatant;
3) exosome extraction: centrifuging the initial supernatant in the step 2) for 5min at the centrifugal force of 300g and the temperature of 4 ℃, discarding the precipitate, centrifuging the supernatant for 10min at the centrifugal force of 1000g and the temperature of 4 ℃, discarding the precipitate, taking the supernatant, adding the extracting solution in the step 1) into the supernatant according to the volume ratio of 1: 0.5, inverting, mixing, incubating at 4 deg.C for 10-20 hr, centrifuging at 4 deg.C under 5000g of centrifugal force for 30min, discarding supernatant, collecting precipitate, suspending with phosphate buffer solution with pH of 7.4, and storing at-80 deg.C.
Example 2
An extraction method of exosomes, comprising the steps of:
1) preparation of PEG extract: weighing 20g of polyethylene glycol with the molecular weight of 5000 and 58.5g of sodium chloride, and fully dissolving the polyethylene glycol and the sodium chloride in pure water to obtain an extracting solution, wherein the concentration of the polyethylene glycol in the extracting solution is 0.2g/L, and the concentration of the sodium chloride is 1 mol/L;
2) cell culture: when the cell fusion degree is about 65%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer solution with the pH value of 7.4, adding a fresh culture medium without serum related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell debris to obtain initial supernatant;
3) exosome extraction: centrifuging the initial supernatant in the step 2) for 8min at the centrifugal force of 500g and the temperature of 20 ℃, discarding the precipitate, centrifuging the supernatant for 20min at the centrifugal force of 1500g and the temperature of 20 ℃, discarding the precipitate, taking the supernatant, adding the extracting solution in the step 1) into the supernatant according to the volume ratio of 1: 1, inverting and fully mixing, incubating at 20 ℃ for 15 hours, centrifuging at 10000g and 20 ℃ for 80min, then discarding the supernatant, collecting the precipitate, suspending with a phosphate buffer solution with pH of 7.4, and storing the sample subgroups at-80 ℃.
Example 3
An extraction method of exosomes, comprising the steps of:
1) preparation of PEG extract: weighing polyethylene glycol with the molecular weight of 6000 and the mass of 30g and sodium chloride with the mass of 87.75g, and fully dissolving in pure water to obtain an extracting solution, wherein the concentration of the polyethylene glycol in the extracting solution is 0.3g/L, and the concentration of the sodium chloride is 1.5 mol/L;
2) cell culture: when the cell fusion degree is about 70%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer solution with the pH value of 7.4, adding a fresh culture medium without serum related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell debris to obtain initial supernatant;
3) exosome extraction: centrifuging the initial supernatant in the step 2) for 8min at the centrifugal force of 600g and the temperature of 30 ℃, discarding the precipitate, centrifuging the supernatant for 20min at the centrifugal force of 1800g and the temperature of 30 ℃, discarding the precipitate, taking the supernatant, adding the extracting solution in the step 1) into the supernatant according to the volume ratio of 1: 1.2 the mixture is inverted and mixed thoroughly, incubated at 30 ℃ for 18 hours, centrifuged at 12000g at 30 ℃ for 100min, the supernatant is discarded, the precipitate is collected, suspended in phosphate buffer at pH 7.4 and the subset of samples is stored at-80 ℃.
Example 4
An extraction method of exosomes, comprising the steps of:
1) preparation of PEG extract: weighing polyethylene glycol with molecular weight of 7000 and mass of 30g and sodium chloride of 87.75g, and fully dissolving in pure water to obtain an extracting solution, wherein the concentration of the polyethylene glycol in the extracting solution is 0.3g/L, and the concentration of the sodium chloride is 1.5 mol/L;
2) cell culture: when the cell fusion degree is about 70%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer solution with the pH value of 7.4, adding a fresh culture medium without serum related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell debris to obtain initial supernatant;
3) exosome extraction: centrifuging the initial supernatant in the step 2) for 10min at the centrifugal force of 700g and the centrifugal force of 37 ℃, discarding the precipitate, centrifuging the supernatant for 40min at the centrifugal force of 1000-2000g and the centrifugal force of 37 ℃, discarding the precipitate, taking the supernatant, adding the extracting solution in the step 1) into the supernatant according to the volume ratio of 1: 1.5 inversion and mixing, at 37 degrees C were incubated for 20 hours, at 15000g centrifugal force, 37 degrees C centrifugal for 150 minutes, then abandon the supernatant, collect the precipitation, with pH 7.4 phosphate buffer suspension, and then the sample subset stored at-80 ℃.
Example 5
An extraction method of exosomes, comprising the steps of:
1) preparation of PEG extract: weighing polyethylene glycol with the molecular weight of 6000 and the mass of 25g and sodium chloride with the mass of 70.2g, and fully dissolving the polyethylene glycol and the sodium chloride in pure water to obtain an extracting solution, wherein the concentration of the polyethylene glycol in the extracting solution is 0.25g/L, and the concentration of the sodium chloride is 1.2 mol/L;
2) cell culture: when the cell fusion degree is about 65%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer solution with the pH value of 7.4, adding a fresh culture medium without serum related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell debris to obtain initial supernatant;
3) exosome extraction: centrifuging the initial supernatant in the step 2) for 10min at the centrifugal force of 700g and the centrifugal force of 37 ℃, discarding the precipitate, centrifuging the supernatant for 40min at the centrifugal force of 1000-2000g and the centrifugal force of 37 ℃, discarding the precipitate, taking the supernatant, adding the extracting solution in the step 1) into the supernatant according to the volume ratio of 1: 1.5 inversion and mixing, at 37 degrees C were incubated for 20 hours, at 15000g centrifugal force, 37 degrees C centrifugal for 150 minutes, then abandon the supernatant, collect the precipitation, with pH 7.4 phosphate buffer suspension, and then the sample subset stored at-80 ℃.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. An extraction method of exosomes, characterized in that the extraction method comprises the following steps:
1) preparation of PEG extract: weighing polyethylene glycol and sodium chloride, and fully dissolving in pure water to obtain an extracting solution;
2) cell culture: when the cell fusion degree is about 60% -70%, removing the original culture medium containing serum, rinsing the cells for three times by using a phosphate buffer, adding a fresh culture medium containing serum-free related components for culturing, continuously culturing until the cell fusion degree reaches 90%, collecting cell culture supernatant, collecting the culture medium, centrifuging, and removing cell fragments to obtain initial supernatant;
3) exosome extraction: centrifuging the initial supernatant in the step 2) twice, taking the supernatant, adding the extracting solution in the step 1), inverting, fully mixing, incubating at 4-37 ℃ for 10-20 hours, centrifuging for the third time, discarding the supernatant, collecting the precipitate, suspending the precipitate with phosphate buffer, and storing the sample subset at-80 ℃.
2. The method for extracting exosomes according to claim 1, wherein in step 1), the molecular weight of polyethylene glycol is 4000-7000, the mass is 10-30g, and the concentration of polyethylene glycol in the extracting solution is 0.1-0.3 g/L.
3. The method for extracting exosomes according to claim 1, wherein in step 1), the mass of sodium chloride is 29.25-87.75g, and the concentration of sodium chloride in the extracting solution is 0.5-1.5 mol/L.
4. The method for extracting exosomes according to claim 1, wherein in step 2) and step 3), the pH of the phosphate buffer is 7.4.
5. Method for extracting exosomes according to claim 1, characterized in that in step 3), said two centrifugation treatments are specifically operated as:
collecting supernatant, centrifuging at 4-37 deg.C for 5-10min under centrifugal force of 300-700g, and removing precipitate;
taking the supernatant, centrifuging at the centrifugal force of 1000-2000g and 4-37 ℃ for 10-40min, and discarding the precipitate.
6. The method for extracting exosomes according to claim 1, wherein in the step 3), the supernatant and the extract extracted after the two centrifugation treatments are mixed according to a volume ratio of 1: 0.5-1.5.
7. The method for extracting exosomes according to claim 1, wherein in the step 3), the operation of the third centrifugation treatment is centrifugal force of 5000-15000g, 4-37 ℃, and centrifugation is performed for 30-150 min.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114921409A (en) * | 2022-07-01 | 2022-08-19 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Preparation method of extracellular vesicles of amniotic fluid mesenchymal stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624557A (en) * | 2018-05-31 | 2018-10-09 | 章毅 | The preparation method and applications of mescenchymal stem cell excretion body |
| CN112048471A (en) * | 2020-09-21 | 2020-12-08 | 淄博市中心医院 | Preparation method of bone marrow mesenchymal stem cell exosome |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108624557A (en) * | 2018-05-31 | 2018-10-09 | 章毅 | The preparation method and applications of mescenchymal stem cell excretion body |
| CN112048471A (en) * | 2020-09-21 | 2020-12-08 | 淄博市中心医院 | Preparation method of bone marrow mesenchymal stem cell exosome |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114921409A (en) * | 2022-07-01 | 2022-08-19 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Preparation method of extracellular vesicles of amniotic fluid mesenchymal stem cells |
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