Extraction method of exosome

Abstract

The invention relates to the field of exosomes, in particular to an extraction method of exosomes. The invention provides an exosome extraction method. The exosome extraction method comprises the following steps: S1, obtaining a preprocessed sample; s2, precipitating the pretreated sample twice, centrifuging, collecting the precipitate, and removing impurities to obtain the exosome, the precipitation adopts a PEG solution with the final concentration of 2-8% w / v; the concentration of the PEG solution in the first precipitation is 2% w / v, and the concentration of the PEG solution in the second precipitation is 8% w / v. The invention provides a high-purity and high-efficiency exosome extraction and purification method, and the method obviously reduces PEG co-precipitation impure protein through ingenious step design, so that the high-purity exosome is obtained while high recovery rate and high efficiency are maintained.

Description

Technical Field

[0001] This invention relates to the field of exosomes, and more particularly to a method for extracting exosomes. Background Technology

[0002] Exosomes are extracellular microvesicles, approximately 30-150 nm in size, with a typical lipid bilayer structure, containing abundant bioactive substances such as proteins, RNA, and lipids. As paracrine products, they are present in cell culture media, plasma, serum, saliva, urine, breast milk, and other biological fluids, playing a vital role in cell communication and significantly influencing surrounding cells and the growth environment. Therefore, exosomes have become a research hotspot in disease diagnosis, prognosis, the development of novel drug delivery systems, and biomedicine.

[0003] Obtaining high-purity, structurally intact exosomes is a prerequisite for downstream functional studies and clinical applications. Currently, there are various methods for exosome extraction and purification. Ultracentrifugation is the traditional gold standard, but it is time-consuming, requires expensive equipment, and may damage the integrity of the exosomes. Size exclusion chromatography and immunocapture methods offer high purity, but are costly and have low throughput. Polymer-based precipitation methods, especially PEG precipitation, have attracted much attention due to their simplicity and low cost. However, traditional PEG precipitation of exosomes also co-precipitates a large amount of lipoprotein or protein polymers in the solution, resulting in low purity exosomes and interfering with subsequent studies. Although some studies have attempted purification by washing or adding density gradient centrifugation after PEG precipitation, these methods have limited effectiveness and increase operational complexity and cost, negating the core advantage of the PEG method's simplicity and speed. Additionally, some studies have used PEG fractionation for exosome purification, but due to the sequential concentration of different fractions, significant exosome loss occurs, resulting in low concentration and purity.

[0004] Therefore, there is an urgent market demand and significant application value in developing an improved process that can effectively overcome the co-precipitation of heterogeneous proteins while retaining the advantages of the PEG method. Summary of the Invention

[0005] In view of this, the present invention provides a method for extracting exosomes. This invention provides a high-purity, high-efficiency method for the extraction and purification of exosomes. Through ingenious step design, this method significantly reduces PEG co-precipitation of impurities, thereby obtaining high-purity exosomes while maintaining high recovery rate and high efficiency.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for extracting exosomes, comprising the following steps:

[0008] S1: Obtain the preprocessed sample;

[0009] S2: The pretreated sample is precipitated twice, centrifuged, the precipitate is collected, impurities are removed, and the exosomes are obtained;

[0010] The precipitate is prepared using a PEG solution with a final concentration of 2-8% w / v;

[0011] The concentration of the PEG solution in the first precipitation was 2% w / v, and the concentration of the PEG solution in the second precipitation was 8% w / v.

[0012] In some embodiments of the present invention, the above extraction method includes the following steps:

[0013] S1: Mix the PEG solution with the sample, incubate, centrifuge for the first time, and collect the supernatant;

[0014] S2: Mix the PEG solution with the supernatant, incubate, centrifuge a second time, and collect the precipitate.

[0015] In some embodiments of the present invention, in the above extraction method, the incubation temperature is 2~8℃ and the time is 2~24h.

[0016] In some embodiments of the present invention, in the above extraction method, the incubation temperature is 4°C and the time is 2h or 24h.

[0017] In some embodiments of the present invention, in the above extraction method, the incubation temperature in S1 is 4°C and the incubation time is 2h; the incubation temperature in S2 is 4°C and the incubation time is 24h.

[0018] In some embodiments of the present invention, in the above extraction method, the conditions for the first centrifugation are 2~8℃, 3000~12000×g, 15~45min; and the conditions for the second centrifugation are 2~8℃, 3000~12000×g, 30~90min.

[0019] In some embodiments of the present invention, in the above extraction method, the conditions for the first centrifugation are 4°C, 5000×g, 20min; and the conditions for the second centrifugation are 4°C, 10000×g, 60min.

[0020] In some embodiments of the present invention, the preprocessing in the above extraction method includes the following steps: removing impurities from the sample to obtain the preprocessed sample.

[0021] In some embodiments of the present invention, the above extraction method employs a two-stage centrifugation process for impurity removal; the first centrifugation is performed at 2-8°C, 1500-5000×g, for 5-15 min; the second centrifugation is performed at 2-8°C, 8000-12000×g, for 10-20 min.

[0022] In some embodiments of the present invention, the above extraction method involves two centrifugation steps for impurity removal; the first centrifugation is performed at 4°C, 3000×g, for 10 min; the second centrifugation is performed at 4°C, 10000×g, for 15 min.

[0023] In some embodiments of the present invention, the above extraction method further includes a purification step before obtaining the exosomes after removing impurities.

[0024] In some embodiments of the present invention, the purification in the above extraction method is carried out using a centrifugal column; the centrifugal column includes a cellulose acetate membrane with a pore size of 0.22~0.45μm; the purification conditions include: 2~8℃, 3000~12000×g, centrifugation for 5~20min.

[0025] In some embodiments of the present invention, in the above extraction method, the centrifuge column comprises a cellulose acetate membrane with a pore size of 0.22 μm; the purification conditions include: 4°C, 3000 × g, centrifugation for 10 min.

[0026] In some embodiments of the present invention, in the above extraction method, the sample is derived from one or more of the following: blood, urine, saliva, effusion, and cell culture medium.

[0027] The present invention also provides exosomes obtained by the above extraction method.

[0028] The beneficial effects of this invention include:

[0029] This invention provides a high-purity and efficient method for exosome extraction and purification. It employs an improved PEG fractionation precipitation method combined with centrifugal column purification, effectively solving the core problem of severe co-precipitation of impurities (in the first precipitation step or when the PEG concentration in the first precipitation step is higher than that in the second) in the traditional PEG method. This improves exosome purity, and the gentle polymer precipitation and relatively mild centrifugation conditions help maintain the integrity and bioactivity of the exosome membrane, making it suitable for downstream exosome functional studies. This method significantly improves exosome purity and activity without increasing operational complexity, while retaining the advantages of the original method: simple operation, low cost, high versatility, and applicability to various biological fluids. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0031] Figure 1 This is a schematic flowchart of a high-purity and high-efficiency exosome extraction and purification method according to the present invention;

[0032] Figure 2 This shows the standard curves for protein content detection of exosomes obtained in Example 1 and Comparative Example 1 of the present invention;

[0033] Figure 3 Electron micrographs of exosomes obtained in Example 1 and Comparative Example 1 of the present invention are shown below;

[0034] Figure 4 This shows the exosome particle size distribution obtained in Example 1 and Comparative Example 1 of the present invention;

[0035] Figure 5 The graph shows the expression results of exosome marker proteins obtained in Example 1 and Comparative Example 1 of the present invention.

[0036] Figure 6 This shows the standard curves for protein content detection of exosomes obtained in Example 2 and Comparative Example 2 of the present invention;

[0037] Figure 7 Electron micrographs of exosomes obtained in Example 2 and Comparative Example 2 of the present invention are shown below;

[0038] Figure 8 This shows the exosome particle size distribution obtained in Example 2 and Comparative Example 2 of the present invention;

[0039] Figure 9 The graph shows the exosomal protein expression results obtained in Example 2 and Comparative Example 2 of the present invention; wherein: A is the expression level of exosomal marker proteins CD81 and TSG101; B is the statistical analysis of marker proteins between Example 2 and Comparative Example 2;

[0040] Figure 10 This shows a standard curve for detecting protein content in exosomes obtained in Example 3 of the present invention;

[0041] Figure 11 The figure shows the protein expression results in the supernatant and total protein of THP-1 cells obtained in Example 3 of the present invention; wherein: A is the IL-1β expression level in the cell supernatant and total protein; B is the statistical analysis of IL-1β expression among different groups. Detailed Implementation

[0042] This invention discloses a method for extracting exosomes.

[0043] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0044] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0045] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0046] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0047] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0048] This invention first provides a precipitation reagent for the extraction and purification of exosomes, and the preparation method is as follows:

[0049] PEG and physiological saline were mixed in a certain proportion to obtain the precipitating reagent for the extraction and purification of exosomes.

[0050] Preferably, the molecular weight of the PEG is 6000~10000, and the concentration is 20%~80%.

[0051] This invention provides a high-purity and efficient method for the extraction and purification of exosomes, comprising the following steps:

[0052] (1) Collect samples of exosomes to be extracted;

[0053] (2) Impurities in the sample are removed by secondary centrifugation to obtain a pretreated sample;

[0054] (3) Add a certain concentration of exosome precipitation reagent to the pretreated sample, mix well, and let stand for incubation;

[0055] (4) After standing, centrifuge to remove precipitated proteins and collect the sample supernatant;

[0056] (5) Add a certain concentration of exosome precipitation reagent to the obtained sample supernatant and let it stand for incubation;

[0057] (6) After standing, centrifuge to obtain exosome precipitate;

[0058] (7) Resuspend the exosome precipitate, centrifuge to remove impurities from the precipitate, and obtain an exosome suspension;

[0059] (8) Transfer the harvested exosome suspension to a centrifuge column and centrifuge to obtain purified exosomes.

[0060] Preferably, in step (2), the first centrifugation conditions are 2~8℃, centrifugation at 1500~5000×g for 5~15min, and the second centrifugation conditions are 2~8℃, centrifugation at 8000~12000×g for 10~20min.

[0061] Preferably, the final concentration of the exosome precipitation reagent in the pretreated sample in step (3) is 1%~10%, and the static incubation conditions are 2~8℃ for 2h~24h.

[0062] Preferably, the centrifugation conditions in step (4) are 2~8℃, 3000~12000×g for 15~45min;

[0063] Preferably, the concentration of the final exosome precipitation reagent in the supernatant of step (5) is 1%~10%, and the static incubation conditions are 2~8℃ for 2h~24h.

[0064] Preferably, the centrifugation conditions in step (6) are 2~8℃, 3000~12000×g for 30~90min;

[0065] Preferably, the resuspending solution of exosome precipitate in step (7) is PBS buffer, and the centrifugation conditions are 2~8℃, 3000~12000×g for 1~5min.

[0066] Preferably, the centrifuge column in step (8) is a cellulose acetate membrane with a pore size of 0.22~0.45μm, and the centrifugation conditions are 2~8℃, centrifugation at 3000~12000 for 5~20min.

[0067] As a further aspect of the present invention: the extraction method can be used for the extraction and purification of exosomes from bodily fluids such as blood, urine, saliva, and effusion, as well as cell culture medium.

[0068] In Examples 1 to 3 and Comparative Examples 1 to 2 of this invention, the raw materials and reagents used can all be purchased from the market.

[0069] In this invention, the concentration of PEG is g / mL.

[0070] The present invention will be further illustrated below with reference to the embodiments:

[0071] Example 1

[0072] The extraction method of this invention is used to extract and purify exosomes from the culture supernatant of mesenchymal stem cells (MSCs). The steps are as follows:

[0073] (1) Preparation of PEG precipitation reagent: Dissolve PEG6000 in physiological saline to a concentration of 40% (w / v).

[0074] (2) Collect 20 mL of serum-free cell culture medium supernatant: When the cell confluence reaches 80-90%, wash twice with PBS buffer to remove residual supernatant, then replace with serum-free culture medium and culture for 48 h before collecting 20 mL of serum-free cell culture medium supernatant.

[0075] (3) Centrifugation to remove impurities: The supernatant from (2) above is placed in a centrifuge at 3000×g and centrifuged at 4℃ for 10min to remove cell debris. The obtained supernatant is then centrifuged at 10000×g and 4℃ for 15min to remove precipitate and obtain cell supernatant.

[0076] (4) Precipitation of impurities: The PEG precipitant obtained in step (1) is slowly added to the cell supernatant in step (3) to a final concentration of 2%, and incubated at 4°C for 2 hours. Then, the supernatant is carefully collected by centrifugation at 5000×g and 4°C for 20 minutes.

[0077] (5) Secondary precipitation of exosomes: PEG precipitation reagent was slowly added to the supernatant obtained in (4) to make the final concentration 8% (w / v), and incubated at 4℃ for 24h. After the incubation, the exosomes were centrifuged at 10000×g at 4℃ for 60min to obtain the exosome precipitate.

[0078] (6) Wash the exosome precipitate: Resuspend the (5) exosome precipitate with PBS buffer, and centrifuge at 12000×g, 4℃ for 2min. Discard the precipitate to obtain a suspension containing exosomes.

[0079] (7) Purification and preservation of exosomes: Transfer the (6) exosome suspension to a 0.22 μm centrifuge column at 3000×g, centrifuge at 4℃ for 10 min, and collect the supernatant after centrifugation to obtain purified exosomes, which can be stored at -80℃ for a long time.

[0080] Comparative Example 1

[0081] Instead of using the exosome extraction method of this invention, exosomes were extracted and purified from the MSC culture supernatant using a single PEG precipitation method before optimization, as follows:

[0082] (1) Preparation of PEG precipitation reagent: Dissolve PEG6000 in physiological saline to a concentration of 40% (w / v).

[0083] (2) Collect 20 mL of serum-free cell culture medium supernatant: When the cell confluence reaches 80-90%, wash twice with PBS buffer to remove residual supernatant, then replace with serum-free culture medium and culture for 48 h before collecting 20 mL of serum-free cell culture medium supernatant.

[0084] (3) Centrifugation to remove impurities: The supernatant from (2) above is placed in a centrifuge at 3000×g and centrifuged at 4℃ for 10min to remove cell debris. The obtained supernatant is then centrifuged at 10000×g and 4℃ for 15min to remove precipitate and obtain cell supernatant.

[0085] (4) Precipitate exosomes: Slowly add the PEG precipitation reagent obtained in step (1) to the cell supernatant in step (3) to make the final concentration 8% (w / v), and incubate at 4℃ for 2h~24h. Then centrifuge at 10000×g at 4℃ for 60min to obtain exosome precipitate.

[0086] (5) Wash the exosome precipitate: Resuspend the (4) exosome precipitate with PBS buffer and centrifuge at 12000×g, 4℃ for 2min. Discard the precipitate to obtain a suspension containing exosomes.

[0087] (6) Purification and preservation of exosomes: Transfer the (5) exosome suspension to a 0.22 μm centrifuge column at 3000×g and centrifuge at 4℃ for 10 min. Collect the supernatant after centrifugation to obtain purified exosomes, which can be stored at -80℃ for a long time.

[0088] The content of exosome proteins extracted using the extraction methods of Example 1 and Comparative Example 1 was detected. The protein concentration results are shown in Table 1, and the standard curve is shown in Table 2. Figure 2 As shown, the two extraction methods yielded high concentrations of exosomes.

[0089] Table 1

[0090]

[0091] The morphology of exosomes extracted using the extraction methods of Example 1 and Comparative Example 1 was examined, and the electron microscopy results are as follows: Figure 3 As shown, the morphology of the ectosomes in the examples and the comparative examples is intact and good, and the examples have fewer impurities and higher purity than the comparative examples.

[0092] The particle size distribution of exosomes extracted using the extraction methods of Example 1 and Comparative Example 1 was determined as follows: Figure 4 As shown, the exosome particle sizes extracted in the examples and comparative examples were 78.8 nm and 142 nm, respectively, both of which are within the exosome particle size range of 30~150 nm. However, the number of particles larger than 150 nm in the examples was significantly less than that in the comparative examples, indicating that there were fewer impurities such as co-precipitated protein aggregates.

[0093] The expression of marker proteins in exosomes extracted using the extraction methods of Example 1 and Comparative Example 1 was detected. The expression results of exosome marker membrane proteins CD81 and TSG101 are as follows: Figure 5 As shown, CD81 and TSG101 protein expression was detected in the exosomes of both Example 1 and Comparative Example 1, and their expression levels were basically the same. However, the expression of CD81 protein showed that the purity of Example 1 was higher than that of Comparative Example 1, indicating the reliability of the exosome extraction method described in this invention.

[0094] Example 2

[0095] The extraction and purification of exosomes from rat plasma using the extraction method of this invention are as follows:

[0096] (1) Preparation of PEG precipitation reagent: Dissolve PEG6000~10000 in physiological saline to a concentration of 20%~80% (w / v).

[0097] (2) Collect 1 mL of rat plasma: Select 200~250g male SD rats, anesthetize the rats, and use a blood collection needle and vacuum blood collection tube to draw blood from the rat abdominal aorta. Immediately divide the collected blood into centrifuge tubes containing anticoagulant, centrifuge at 3000×g for 15 min, carefully aspirate the supernatant, and collect 1 mL of rat plasma.

[0098] (3) Centrifugation to remove impurities: 1 mL of rat plasma collected in (2) above was placed in a centrifuge at 3000×g and centrifuged at 4℃ for 10 min. The precipitate was discarded, and the supernatant was collected and transferred to a new centrifuge tube. The supernatant was then centrifuged at 10000×g and 4℃ for 15 min. The precipitate was discarded, and the supernatant was collected in a new centrifuge tube to obtain a pretreated rat plasma sample.

[0099] (4) Dilute rat plasma samples: Add 3 mL of PBS buffer to the rat plasma sample in (3) above.

[0100] (5) Precipitation of impurities: Precipitation of exosomes: The PEG precipitation reagent obtained in step (1) is slowly added to the rat plasma sample diluted in (4) above to make the final concentration 2%, and incubated at 4°C for 2 hours. Then, the sample is centrifuged at 5000×g at 4°C for 20 minutes and the supernatant is carefully collected.

[0101] (6) Secondary precipitation of exosomes: Secondary precipitation of exosomes: Slowly add the PEG precipitation reagent obtained in step (1) to the supernatant obtained in (5) to make the final concentration 8% (w / v), and incubate at 4℃ for 24h. After the incubation, centrifuge at 10000×g at 4℃ for 60min to obtain exosome precipitate.

[0102] (7) Wash the exosome precipitate: Resuspend the (6) exosome precipitate with PBS buffer, and centrifuge at 12000×g, 4℃ for 2min. Discard the precipitate to obtain a suspension containing exosomes.

[0103] (8) Purification and preservation of exosomes: Transfer the (7) exosome suspension to a 0.2 μm centrifuge column at 3000×g, centrifuge at 4℃ for 10 min, and collect the supernatant after centrifugation to obtain purified exosomes, which can be stored at -80℃ for a long time.

[0104] Comparative Example 2

[0105] Instead of using the exosome extraction method of this invention, exosomes were extracted and purified from rat plasma using a pre-optimized two-stage PEG precipitation method, as follows:

[0106] (1) Preparation of PEG precipitation reagent: Dissolve PEG6000 in physiological saline to a concentration of 40% (w / v).

[0107] (2) Collect 1 mL of rat plasma: Select 200-250g male SD rats. After anesthetizing the rats, use a blood collection needle and a vacuum blood collection tube to draw blood from the rat's abdominal aorta. Immediately divide the collected blood into centrifuge tubes containing anticoagulant, centrifuge at 3000×g for 15 min, carefully aspirate the supernatant, and collect 1 mL of rat plasma.

[0108] (3) Centrifugation to remove impurities: 1 mL of rat plasma collected in (2) above was placed in a centrifuge at 3000×g and centrifuged at 4℃ for 10 min. The precipitate was discarded, and the supernatant was collected and transferred to a new centrifuge tube. The supernatant was then centrifuged at 10000×g and 4℃ for 15 min. The precipitate was discarded, and the supernatant was collected in a new centrifuge tube to obtain a pretreated rat plasma sample.

[0109] (4) Dilute rat plasma samples: Add 3 mL of PBS buffer to the rat plasma sample in (3) above.

[0110] (5) First precipitation of exosomes: The PEG precipitation reagent obtained in step (1) is slowly added to the rat plasma sample diluted in (4) above to make the final concentration 8% (w / v), and incubated at 4℃ for 24h. Then, it is centrifuged at 10000×g and 4℃ for 60min to obtain crude exosome precipitate.

[0111] (6) Secondary precipitation of exosomes: The crude exosome precipitate of (5) was resuspended in PBS buffer and the PEG precipitation reagent obtained in step (1) was slowly added to make the final concentration 5% (w / v). The mixture was incubated at 4℃ for 2h. After the incubation, the mixture was centrifuged at 10000×g at 4℃ for 60min to obtain the exosome precipitate again.

[0112] (7) Wash the exosome precipitate: Resuspend the (6) exosome precipitate with PBS buffer, and centrifuge at 12000×g, 4℃ for 2min. Discard the precipitate to obtain a suspension containing exosomes.

[0113] (8) Purification and preservation of exosomes: Transfer the (7) exosome suspension to a 0.22 μm centrifuge column at 3000×g, centrifuge at 4℃ for 10 min, and collect the supernatant after centrifugation to obtain purified exosomes, which can be stored at -80℃ for a long time.

[0114] The content of exosome proteins extracted using the extraction methods of Example 2 and Comparative Example 2 was detected. The protein concentration results are shown in Table 2, and the standard curve is shown in Table 2. Figure 6 As shown, both extraction methods yielded high concentrations of exosomes.

[0115] Table 2

[0116]

[0117] The morphology of exosomes extracted using the extraction methods of Example 2 and Comparative Example 2 was examined, and the electron microscopy results are as follows: Figure 7 As shown, the exosomes in Example 2 and Comparative Example 2 have intact and good morphology, and Example 2 has fewer impurities and higher purity than Comparative Example 2.

[0118] The exosomes extracted using the extraction methods of Example 2 and Comparative Example 2 were subjected to particle size analysis, and the particle size distribution results are as follows: Figure 8 As shown, the exosomes extracted in Example 2 and Comparative Example 2 had particle sizes of 91.3 nm and 122 nm, respectively, both of which are within the exosome particle size range of 30~150 nm. However, in Example 2, the number of particles within the particle size range was more concentrated and the particle size was more uniform, indicating that there were fewer co-precipitated impurities such as proteins.

[0119] The expression of marker proteins in exosomes extracted using the extraction methods of Example 2 and Comparative Example 2 was detected. The expression results of exosome marker membrane proteins CD81 and TSG101 are as follows: Figure 9 As shown, CD81 and TSG101 protein expression was detected in the exosomes of both Example 2 and Comparative Example 2. Moreover, the expression level of Example 2 was significantly higher than that of Comparative Example 2, and there was a significant statistical significance. This indicates that the extraction method of the present invention can extract more exosomes and reduce the loss of exosomes, further demonstrating that the exosome extraction method of the present invention has good reliability.

[0120] Example 3

[0121] The MSCs exosome-2 (Exo-2) extracted in Example 1 and the MSCs exosome-1 (Exo-1) extracted in Comparative Example 1 were incubated with the same amount of protein in a macrophage inflammation model to verify the anti-inflammatory effect of MSCs-derived exosomes. The steps are as follows:

[0122] (1) Differentiation of human monocytic leukemia cells (THP-1): When the THP-1 cell confluence reaches more than 90%, the cells are seeded in 6-well plates and stimulated to differentiate into macrophages using phosphatidylinositol (PMA) at a final concentration of 30 ng / mL.

[0123] (2) Pretreatment of cells with MSCs-derived exosomes: After THP-1 cells adhered to the wells of the 6-well plate, the following groups were set up: normal group (N), inflammation model group (LPS / ATP), inflammation model + exosome-1 group (LPS / ATP-Exo-1), and inflammation model + exosome-2 group (LPS / ATP-Exo-2). The latter two groups were incubated with 100 μg of Exo-1 and Exo-2 at a rate of 100 μg / well, respectively, while the other two groups were incubated with an equal amount of PBS.

[0124] (3) Establish LPS / ATP-induced macrophage inflammation model: After incubation for 24 hours as described in (2), except for the normal group, the other groups were stimulated with a final concentration of 1 μg / mL LPS for 4 hours and 30 mM ATP for 30 minutes. Then, the cell supernatant and total protein of all groups were collected.

[0125] The total protein content of THP-1 cells extracted in Example 3 was detected, and the protein concentration results are shown in Table 3, along with the standard curve shown. Figure 10 As shown:

[0126] Table 3

[0127]

[0128] The inflammatory factors were detected in the THP-1 cell supernatant and total protein extracted in Example 3 above, such as... Figure 11 As shown, the MSCs-derived exosomes Exo-2 obtained by the extraction method of this invention can significantly reduce the expression of the pro-inflammatory cytokine IL-1β in the supernatant and total protein of THP-1 cells, inhibit the inflammatory response, and have good statistical significance. This indicates that the exosome extraction method of this invention is suitable for downstream function research of exosomes, maintains good exosome properties, and does not affect exosome activity.

[0129] The present invention proposes a high-purity and high-efficiency method for the extraction and purification of exosomes. It adopts PEG fractionation precipitation combined with centrifugal column purification method, which is simple to operate, saves time and effort, and does not require expensive equipment. The extracted exosomes have a large content and high purity, and can be used for a series of downstream functional experiments of exosomes.

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting exosomes, characterized in that, Includes the following steps: S1: Obtain the preprocessed sample; S2: The pretreated sample is precipitated twice, centrifuged, the precipitate is collected, impurities are removed, and the exosomes are obtained; The precipitate is prepared using a PEG solution with a final concentration of 2-8% w / v; The concentration of the PEG solution in the first precipitation was 2% w / v, and the concentration of the PEG solution in the second precipitation was 8% w / v.

2. The extraction method as described in claim 1, characterized in that, The two precipitation processes include the following steps: S1: Mix the PEG solution with the sample, incubate, centrifuge for the first time, and collect the supernatant; S2: Mix the PEG solution with the supernatant, incubate, centrifuge a second time, and collect the precipitate.

3. The extraction method as described in claim 2, characterized in that, The incubation temperature is 2~8℃, and the time is 2~24h.

4. The extraction method as described in claim 3, characterized in that, The conditions for the first centrifugation are 2~8℃, 3000~12000×g, 15~45min; the conditions for the second centrifugation are 2~8℃, 3000~12000×g, 30~90min.

5. The extraction method as described in claim 4, characterized in that, The preprocessing includes the following steps: removing impurities from the sample to obtain the preprocessed sample.

6. The extraction method as described in claim 5, characterized in that, The impurity removal process involves two centrifugation steps; the first centrifugation is performed at 2-8°C, 1500-5000×g, for 5-15 min; the second centrifugation is performed at 2-8°C, 8000-12000×g, for 10-20 min.

7. The extraction method as described in claim 6, characterized in that, The process of removing impurities and obtaining the exosomes also includes a purification step.

8. The extraction method as described in claim 7, characterized in that, The samples were derived from one or more of the following: blood, urine, saliva, effusion, and cell culture medium.

9. Exosomes obtained by the extraction method according to any one of claims 1 to 8.

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