Method for extracting extracellular vesicles from induced sputum

Abstract

The invention relates to the technical field of cell separation and extraction, and provides a method for extracting extracellular vesicles from induced sputum. Comprising the following steps: S1, atomizing and collecting induced sputum, picking out sputum plugs, and adding an isotonic phosphate buffer solution which contains 0.1 wt% of drocisteine and 0.1 wt% of tween-20 and has the pH value of 6.0 for dissolving, so as to obtain an induced sputum dissolving solution rich in extracellular vesicles; wherein the dissolving temperature is 37 + / -0.1 DEG C, and digestion is performed for 15 + / -1 min under the condition that the rotating speed of a shaking table is 50 + / -1 rpm; and S2, extracting the extracellular vesicles from the induced sputum dissolving solution through a negative pressure size exclusion chromatography method. According to the invention, the multi-component dissolving agent of the hydroxymethylsteine and the Tween-20 is adopted to promote the dissolving of sputum and protect protein and nucleic acid in extracellular vesicles from being degraded; the negative pressure size exclusion chromatography can accurately control the flow velocity driven by negative pressure, ensures stable migration of extracellular vesicles in a chromatographic column, and avoids vesicle rupture caused by too fast flow velocity or low separation efficiency caused by too slow flow velocity.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell separation and extraction, and particularly relates to a method for extracting extracellular vesicles from induced sputum. Background Art

[0002] Induced sputum is a safe and effective method for obtaining clinical samples, which is widely used in basic research and clinical applications for diagnosing and monitoring the status of lung diseases, especially conditions related to chronic lung diseases such as asthma, chronic obstructive pulmonary disease, and interstitial lung disease. The advantage of this technique is that it can sample the airway in a non-invasive manner, which is particularly important for the examination of patients with severe respiratory diseases, avoiding the risks brought by bronchoscopy to patients.

[0003] Extracellular vesicles (EVs) refer to nanoscale lipid bilayer vesicles released by almost all cells during activation, injury, or apoptosis. They are widely distributed in human tissue fluids, including blood, lymph, cerebrospinal fluid, urine, saliva, milk, and ascites. Due to the complexity and heterogeneity of the biological sources of extracellular vesicles, vesicle size is the most commonly used parameter to distinguish extracellular vesicle types. According to the particle size, extracellular vesicles can be divided into three categories, namely exosomes (diameter 40 - 100nm), microvesicles (diameter 100 - 1000nm), and apoptotic bodies (diameter 1000 - 5000nm). Extracellular vesicles have a transport function and mediate cell - to - cell communication by delivering different substances (including proteins, DNA, microRNA, lncRNA, circRNA, and mRNA) to target cells. After being endocytosed into cells, extracellular vesicles can release their inherent components through three pathways, especially by fusing with the cytoplasmic membrane, fusing with the endoplasmic reticulum, and fusing with the endoplasmic membrane or endoplasmic membrane - ruptured materials. Extracellular vesicles can participate in cell - to - cell communication in the lungs and regulate pulmonary pathophysiological processes. There is also evidence that extracellular vesicles are related to the development of chronic inflammatory respiratory diseases.

[0004] In clinical research, serum and urine are commonly used samples for the isolation, enrichment, and biomarker screening of extracellular vesicles. There have been numerous clinical studies based on serum and urine extracellular vesicles, demonstrating the crucial role of circulating extracellular vesicles in disease diagnosis and prognosis. The entire process of induced sputum extracellular vesicle extraction includes clinical sample collection and preservation, sputum dissolution, extraction and enrichment of extracellular vesicles from the dissolution solution, and verification of the morphology and protein molecular level of extracellular vesicles. Research has shown that the functions of extracellular vesicles in physiological and pathological states may be affected by the external environment, including the composition and pH value of the dissolution solution. The common dissolution reagent DTT can release extracellular vesicles by disrupting the sputum protein polymer network. However, in the subsequent process, smaller protein polymer fibers may be co-isolated with extracellular vesicles, resulting in the presence of protein polymer fiber contaminants in the extracellular vesicle sample, which reduces the purity of extracellular vesicles and affects the accuracy of subsequent experimental results such as proteomics analysis. Currently, in the extraction of extracellular vesicles after sputum dissolution, differential centrifugation or kit PEG precipitation methods are mostly used. The differential centrifugation method is time-consuming (3 - 4 hours), consumes a large amount of samples, causes obvious vesicle damage, and has a low recovery rate. The PEG precipitation method often introduces other protein contaminants from the enzymatic digestion solution. Therefore, how to quickly extract and enrich high-purity extracellular vesicles from sputum remains an urgent problem to be solved. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for extracting extracellular vesicles from induced sputum, aiming to solve the problems mentioned in the background art.

[0006] The present invention provides a method for extracting extracellular vesicles from induced sputum, comprising the following steps: Step S1: After atomizing and collecting induced sputum, pick out the sputum plugs, and add an isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 for dissolution to obtain an induced sputum dissolution solution rich in extracellular vesicles. The dissolution temperature is 37 ± 0.1 °C, and digestion is carried out for 15 ± 1 min under the condition that the shaker speed is 50 ± 1 rpm; Step S2: Extract extracellular vesicles from the induced sputum dissolution solution by negative pressure size exclusion chromatography.

[0007] Further, in step S1, after atomizing and collecting induced sputum, the specific steps for picking out the sputum plugs are as follows: Obtain the patient's induced sputum sample by atomizing concentrated brine; weigh the centrifuge tube, pick out the sputum plugs with forceps, put them into the centrifuge tube, and weigh again to determine the mass of the sputum plugs.

[0008] Further, in step S1, an isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 is added to the centrifuge tube; then the centrifuge tube is placed horizontally on a shaker for dissolution; after the dissolution is completed, the centrifuge tube is centrifuged to obtain the first supernatant, which is the induced sputum lysate rich in extracellular vesicles with cells removed, where the centrifugation conditions are 3000 rpm, 4 ± 0.1 °C, and 5 min.

[0009] Further, when the mass of the sputum plug is 2 ± 0.5 g, the addition amount of the isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 is 10 mL.

[0010] Further, in step S2, the extracellular vesicles are extracted from the induced sputum lysate by negative pressure size exclusion chromatography, and the specific steps are as follows: Step S21: The induced sputum lysate is centrifuged to obtain the second supernatant, where the centrifugation conditions are 17000 g, 4 ± 0.1 °C, and 30 ± 1 min, and the second supernatant is the secondary supernatant; Step S22: After the second supernatant is filtered through a 0.22 μm filter, the obtained filtrate is added to a negative pressure size exclusion chromatography column; Step S23: After the filtrate completely enters the negative pressure size exclusion chromatography column, a phosphate buffer solution is added; the phosphate buffer solution is added in 6 - 8 times; the fractions generated after the first 1 - 2 times and the last time of adding the phosphate buffer solution are emptied, and all the fractions generated after other additions of the phosphate buffer solution are collected to obtain an extracellular vesicle suspension.

[0011] Further, in step S22, the negative pressure size exclusion chromatography column is filled with 10 ml of agarose gel CL-6B.

[0012] Further, in step S22, the constant negative pressure of the negative pressure size exclusion chromatography column is 1 - 100 μL / s.

[0013] Further, in step S23, the addition amount of the phosphate buffer solution each time is 500 μL - 1 mL, and the collection amount of the extracellular vesicle suspension is 5 times the addition amount of the phosphate buffer solution each time.

[0014] Further, in step S23, after obtaining the extracellular vesicle suspension, the extracellular vesicle suspension is added to an ultrafiltration centrifuge tube and centrifuged until the solid phase is 8 - 15% of the total amount of the extracellular vesicle suspension, and the obtained solid phase is the concentrated induced sputum extracellular vesicle suspension, where the centrifugation conditions are 4000 g, 4 ± 0.1 °C, and 15 ± 1 min.

[0015] The present invention has the following beneficial effects: (1) Use a multi-component solvent of ambroxol and Tween-20 to promote sputum dissolution and protect the proteins and nucleic acids in extracellular vesicles from degradation; negative pressure size exclusion chromatography can precisely control the flow rate driven by negative pressure to ensure the stable migration of extracellular vesicles in the chromatographic column and avoid vesicle rupture caused by too fast flow rate or low separation efficiency caused by too slow flow rate.

[0016] (2) Compared with the differential centrifugation method, this method can be quickly implemented in clinical laboratories, with less sample consumption, high recovery rate, shortened detection cycle (1-2 hours), improved diagnosis and treatment efficiency; it provides more efficient, simple, economical and reliable technical support for biomedical research. Brief Description of the Drawings

[0017] By referring to the following drawings, the exemplary embodiments of the present invention can be more completely understood: Figure 1 It is a flow chart of the method for extracting extracellular vesicles from induced sputum in Example 1 of the present invention.

[0018] Figure 2 It is a graph of the number of extracellular vesicle particles and an electron micrograph of extracellular vesicles collected by the negative pressure size exclusion chromatography column in Example 1 of the present invention; wherein: Figure 2 A in it is the graph of the number of extracellular vesicle particles; Figure 2 B in it is the electron micrograph of extracellular vesicles, scale bar: 100 nm.

[0019] Figure 3 It is an electron micrograph of the induced sputum extracellular vesicle suspension in Example 1 and Comparative Example 1 of the present invention, scale bar: 1.0 μm.

[0020] Figure 4 Particle size graphs of the induced sputum extracellular vesicle suspensions in Example 1 and Comparative Example 1 of the present invention; wherein: Figure 4 A in it is the particle size distribution curve graph of Example 1; Figure 4 B in it is the particle size distribution curve graph of Comparative Example 1.

[0021] Figure 5 Immunoblot identification graphs of extracellular vesicle marker proteins in Example 1 and Comparative Example 1 of the present invention.

[0022] Figure 6 It is a graph of the extraction amount of extracellular vesicles in Example 1 and Comparative Example 1 of the present invention. Detailed Embodiments

[0023] To make the technical problems, technical solutions, and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and do not limit the present invention.

[0025] The embodiment of the present invention provides a method for extracting extracellular vesicles from induced sputum, including the following steps: Step S1: After atomizing and collecting induced sputum, pick out the sputum plugs, add an isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol and 0.1% vol Tween-20 for dissolution to obtain an induced sputum dissolution solution rich in extracellular vesicles; the temperature of dissolution is 37 ± 0.1 °C, and digestion is carried out for 15 ± 1 min under the condition that the shaker speed is 50 ± 1 rpm; Step S2: Extract extracellular vesicles from the induced sputum dissolution solution by negative pressure size exclusion chromatography.

[0026] Specifically, in step S1, after atomizing and collecting induced sputum, the steps of picking out the sputum plugs are as follows: Obtain a patient's induced sputum sample by atomizing concentrated saline; weigh the centrifuge tube, pick out the sputum plugs with forceps, put them into the centrifuge tube, and weigh again to determine the mass of the sputum plugs.

[0027] Specifically, in step S1, add an isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol and 0.1% vol Tween-20 to the centrifuge tube; then place the centrifuge tube horizontally on the shaker for dissolution; after completion of dissolution, centrifuge the centrifuge tube to obtain a first supernatant, which is the induced sputum dissolution solution rich in extracellular vesicles after removing cells, and the centrifugation conditions are 3000 rpm, 4 ± 0.1 °C, and 5 min.

[0028] Specifically, when the mass of the sputum plugs is 2 ± 0.5 g, the addition amount of the isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol and 0.1% vol Tween-20 is 10 mL.

[0029] Specifically, in step S2, the steps of extracting extracellular vesicles from the induced sputum dissolution solution by negative pressure size exclusion chromatography are as follows: Step S21: Centrifuge the induced sputum dissolution solution to obtain a second supernatant, and the centrifugation conditions are 17000 g, 4 ± 0.1 °C, and 30 ± 1 min, and the second supernatant is the secondary supernatant; Step S22: After filtering the second supernatant through a 0.22 μm filter, the obtained filtrate is added to a negative pressure size exclusion chromatography column; Step S23: After the filtrate completely enters the negative pressure size exclusion chromatography column, phosphate buffer solution is added; the phosphate buffer solution is added in 6 - 8 times; the fractions generated after the first 1 - 2 times and the last time of adding the phosphate buffer solution are emptied, and all the fractions generated after other additions of the phosphate buffer solution are collected to obtain an extracellular vesicle suspension.

[0030] Specifically, in step S22, the negative pressure size exclusion chromatography column is packed with 10 ml of agarose gel CL - 6B.

[0031] Specifically, in step S22, the constant negative pressure of the negative pressure size exclusion chromatography column is 1 - 100 μL / s.

[0032] Specifically, in step S23, the addition amount of the phosphate buffer solution each time is 500 μL - 1 mL, and the collection amount of the extracellular vesicle suspension is 5 times the addition amount of the phosphate buffer solution each time.

[0033] Specifically, in step S23, after obtaining the extracellular vesicle suspension, the extracellular vesicle suspension is added to an ultrafiltration centrifuge tube for centrifugation until the solid phase is 8 - 15% of the total amount of the extracellular vesicle suspension, and the obtained solid phase is a concentrated induced sputum extracellular vesicle suspension, where the centrifugation conditions are 4000 g, 4 ± 0.1 °C, and 15 ± 1 min.

[0034] Reagents: Multicomponent solubilizer: 0.1 g of ambroxol and 1 mL of Tween - 20 are dissolved in 100 mL of pH = 6.0 isotonic phosphate buffer solution and stored in aliquots at - 80 °C; pH = 6.0 isotonic phosphate buffer solution: 8.01 g of NaCl, 0.20 g of KCl, 1.78 g of Na2HPO4·2H2O, and 0.27 g of KH2PO4 are added to 900 mL of sterile double - distilled water, the pH is adjusted to 6.0 using hydrochloric acid, and the volume is made up to 1 L.

[0035] Apparatus: Negative pressure size exclusion chromatography column: The outlet is connected to a collection bottle through a Luer connector. The collection bottle has a vent at the bottle neck, and the vent can be connected to a vacuum pump. After connecting the vent to the pump, a constant negative pressure can be provided to the size exclusion chromatography column; after use, the negative pressure size exclusion chromatography column is washed with one column bed volume of phosphate buffer solution. After washing, it can continue to be used for the extraction of extracellular vesicles from the next sample, or it can be stored in a 4 °C refrigerator after adding 20% ethanol.

[0036] Example 1: (1)After atomizing and collecting induced sputum, pick out the sputum plugs with a mass of 2 ± 0.5 g and place them in a 15 mL centrifuge tube. Then add 10 mL of isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 to the centrifuge tube. Place the 15 mL centrifuge tube horizontally on a water bath shaker for dissolution. The dissolution temperature is 37 °C, and the shaker speed is 50 rpm for 15 min. After dissolution, centrifuge the 15 mL centrifuge tube at 3000 rpm with a centrifuge temperature of 4 °C for 5 min. The obtained supernatant is the induced sputum lysate with cells removed. Transfer the supernatant to a new 15 mL centrifuge tube. (2)Centrifuge the induced sputum lysate at 17000 rpm with a centrifuge temperature of 4 °C for 30 min to obtain a secondary supernatant. Filter the secondary supernatant through a 0.22 μm filter, and add the obtained filtrate to a negative pressure size exclusion chromatography column. The negative pressure size exclusion chromatography column is filled with 10 ml of Sepharose CL-6B, and the amount of the secondary supernatant used is 1 ml. After the filtrate completely enters the negative pressure size exclusion chromatography column, add phosphate buffer solution (PBS) in 8 portions, with each portion being 500 μl. The fractions generated after the first 1-2 and the last addition of the phosphate buffer solution are emptied, and collect the fractions generated after the 3-7th addition of the heated phosphate buffer solution to obtain 2.5 mL of extracellular vesicle suspension. (3)Add 2.5 mL of extracellular vesicle suspension to an ultrafiltration centrifuge tube (100KDa / 4ml), centrifuge at 4000g with a centrifuge temperature of 4 °C for 5 min, and centrifuge until the solid phase is 10% of the total amount of the extracellular vesicle suspension. The obtained solid phase is the concentrated induced sputum extracellular vesicle suspension (EVs suspension), which can be stored at -80 °C for a long time for subsequent protein and RNA detection.

[0037] The flowcharts of steps (1), (2), and (3) in Example 1 are as Figure 1 shown, and the whole process takes 1 - 2 hours.

[0038] The number of extracellular vesicle particles and the electron microscopy results of extracellular vesicles in the fractions collected by the negative pressure size exclusion chromatography column with the phosphate buffer solution added in portions are as Figure 2 shown. The results show that the 3-7th fractions meet the requirements. Therefore, in step (2), collect the fractions generated after the 3-7th addition of the heated phosphate buffer solution to obtain 2.5 mL of extracellular vesicle suspension, which has a high recovery rate and can be used for subsequent protein and RNA detection.

[0039] Comparative Example 1: Referring to Example 1, the difference is that: 10 mL of isosmotic phosphate buffer with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 is replaced by 10 mL of 0.1% DTT.

[0040] Comparative Example 2: Referring to Example 1, the difference is that: 10 mL of isosmotic phosphate buffer with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 is replaced by 10 mL of 0.1% trypsin.

[0041] Result analysis: The electron microscopy results of the induced sputum extracellular vesicle suspension in Example 1 and Comparative Example 1 are as Figure 3 shown; the particle size results of the induced sputum extracellular vesicle suspension in Example 1 and Comparative Example 1 are as Figure 4 shown; the immunoblot identification results of the extracellular vesicle marker proteins in Example 1 and Comparative Example 1 are as Figure 5 shown; the extraction amount of extracellular vesicles in Example 1 and Comparative Example 1 is as Figure 6 shown. Figure 6 In this, Dissolution Solution 1 is isosmotic phosphate buffer with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20, and Dissolution Solution 2 is 0.1% DTT.

[0042] The results show that: on the premise that the sputum source, sampling weight, and volume are the same, compared with dissolution with 0.1% DTT, dissolution with isosmotic phosphate buffer with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 can reduce protein polymer fiber contaminants (as Figure 3 shown); on the premise that the sputum source, sampling weight, and volume are the same, compared with dissolution with 0.1% DTT, dissolution with isosmotic phosphate buffer with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 can inhibit the rupture of extracellular vesicles and obtain more extracellular vesicles (as Figure 4 and Figure 5 shown), that is, compared with Comparative Example 1, the extracellular vesicle marker proteins are more enriched in the induced sputum extracellular vesicle suspension extracted in Example 1, and the extracellular vesicle suspension is qualified in terms of protein level quality.

[0043] In summary, the present invention uses a multi-component solvent of ambroxol and Tween-20 to promote sputum dissolution and protect the proteins and nucleic acids in extracellular vesicles from degradation; negative pressure size exclusion chromatography can precisely control the flow rate driven by negative pressure to ensure the stable migration of extracellular vesicles in the chromatographic column and avoid vesicle rupture caused by too fast flow rate or low separation efficiency caused by too slow flow rate. Compared with the differential centrifugation method, this method can be quickly implemented in a clinical laboratory, with less sample consumption, high recovery rate, shortened detection cycle (1-2 hours), and improved diagnosis and treatment efficiency; it provides more efficient, simple, economical and reliable technical support for biomedical research.

[0044] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting extracellular vesicles from induced sputum, characterized in that, It includes the following steps: Step S1: After atomizing and collecting induced sputum, pick out the sputum plugs, add an isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 for dissolution to obtain an induced sputum dissolution solution rich in extracellular vesicles; the dissolution temperature is 37 ± 0.1 °C, and digestion is carried out for 15 ± 1 min under the condition that the shaker speed is 50 ± 1 rpm; Step S2: The induced sputum dissolution solution is used to extract extracellular vesicles by negative pressure size exclusion chromatography.

2. The method for extracting extracellular vesicles from induced sputum according to claim 1, wherein: In Step S1, after atomizing and collecting induced sputum, pick out the sputum plugs. The specific steps are as follows: Obtain the patient's induced sputum sample by atomizing concentrated saline; weigh the centrifuge tube, pick out the sputum plugs with forceps, put them into the centrifuge tube, and weigh again to determine the mass of the sputum plugs.

3. The method for extracting extracellular vesicles from induced sputum according to claim 2, wherein: In Step S1, add an isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 to the centrifuge tube; then place the centrifuge tube horizontally on the shaker for dissolution; after completion of dissolution, centrifuge the centrifuge tube to obtain the first supernatant, which is the induced sputum dissolution solution rich in extracellular vesicles after removing cells, where the centrifugation conditions are 3000 rpm, 4 ± 0.1 °C, and 5 min.

4. The method for extracting extracellular vesicles from induced sputum according to claim 3, wherein: When the mass of the sputum plugs is 2 ± 0.5 g, the addition amount of the isosmotic phosphate buffer solution with a pH of 6.0 containing 0.1% wt ambroxol hydrochloride and 0.1% vol Tween-20 is 10 mL.

5. The method for extracting extracellular vesicles from induced sputum according to claim 4, characterized in that: In Step S2, the induced sputum dissolution solution is used to extract extracellular vesicles by negative pressure size exclusion chromatography. The specific steps are as follows: Step S21: Centrifuge the induced sputum dissolution solution to obtain the second supernatant, where the centrifugation conditions are 17000 g, 4 ± 0.1 °C, and 30 ± 1 min, and the second supernatant is the secondary supernatant; Step S22: After filtering the second supernatant through a 0.22 μm filter, add the obtained filtrate to a negative pressure size exclusion chromatography column; Step S23: After the filtrate completely enters the negative pressure size exclusion chromatography column, add a phosphate buffer solution; Among them, the phosphate buffer solution is added in 6 - 8 times; the fractions generated after the first 1 - 2 times and the last time of adding the phosphate buffer solution are emptied, and all the fractions generated after other additions of the phosphate buffer solution are collected to obtain an extracellular vesicle suspension.

6. The method for extracting extracellular vesicles from induced sputum according to claim 5, characterized in that: In Step S22, the negative pressure size exclusion chromatography column is filled with 10 ml of agarose gel CL-6B.

7. The method for extracting extracellular vesicles from induced sputum according to claim 6, characterized in that: In Step S22, the constant negative pressure of the negative pressure size exclusion chromatography column is 1 - 100 μL / s.

8. The method for extracting extracellular vesicles from induced sputum according to claim 7, wherein: In Step S23, the addition amount of each phosphate buffer solution is 500 μL - 1 mL, and the collection amount of the extracellular vesicle suspension is 5 times the addition amount of each phosphate buffer solution.

9. The method for extracting extracellular vesicles from induced sputum according to claim 8, characterized in that: In Step S23, after obtaining the extracellular vesicle suspension, add the extracellular vesicle suspension to an ultrafiltration centrifuge tube for centrifugation until the solid phase is 8 - 15% of the total amount of the extracellular vesicle suspension to obtain a concentrated induced sputum extracellular vesicle suspension, where the centrifugation conditions are 4000 g, 4 ± 0.1 °C, and 15 ± 1 min.

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