A method for extracting extracellular vesicles from induced sputum

Abstract

The present invention relates to the field of cell separation and extraction technology, and provides a method for extracting extracellular vesicles from induced sputum. Comprise the following steps: step S1: after atomization collection of induced sputum, pick out sputum plug, add pH=6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% Tween 20 for dissolution, and obtain an induced sputum lysate rich in extracellular vesicles; wherein the dissolution temperature is 37±0.1°C, and the shaking table speed is 50±1rpm for digestion 15±1min; step S2: the induced sputum lysate extracts extracellular vesicles by negative pressure size exclusion chromatography. The present invention adopts a multi-component solubilizer of hydroxymethylcysteine ​​and Tween 20 to promote sputum dissolution and protect proteins and nucleic acids in extracellular vesicles from being degraded; negative pressure size exclusion chromatography can accurately control the flow rate driven by negative pressure, ensure that extracellular vesicles migrate stably in the chromatographic column, avoid vesicle rupture caused by too fast flow rate or low separation efficiency caused by too slow flow rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell separation and extraction, and in particular 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, widely used in basic research and clinical applications for diagnosing and monitoring lung disease status, particularly those associated with chronic lung conditions such as asthma, chronic obstructive pulmonary disease, and interstitial lung disease. The advantage of this technique is that it allows for non-invasive sampling of the airways. This is particularly important for examining patients with severe respiratory conditions, avoiding the risks associated with bronchoscopy.

[0003] Extracellular vesicles (EVs) are nanometer-sized lipid bilayer vesicles released by virtually all cells upon 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 their biological origins, vesicle size is the most commonly used parameter to distinguish EV types. Based on their size, EVs can be divided into three categories: exosomes (40-100 nm in diameter), microvesicles (100-1000 nm in diameter), and apoptotic vesicles (1000-5000 nm in diameter). EVs have transport functions, mediating intercellular communication by delivering diverse cargoes (including proteins, DNA, microRNAs, lncRNAs, circRNAs, and mRNAs) to target cells. After endocytosis, EVs can release their contents through three pathways: fusion with the plasma membrane, fusion with the endoplasmic reticulum, and fusion with the endoplasmic membrane or ruptured endoplasmic membrane material. Extracellular vesicles can participate in intercellular communication in the lungs and regulate lung 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 extracellular vesicle (EV) isolation, enrichment, and marker screening. Numerous clinical studies based on serum and urine EVs have demonstrated the critical role of circulating EVs in disease diagnosis and prognosis. The entire induced sputum EV extraction process includes clinical sample collection and preservation, sputum lysis, EV extraction and enrichment from the lysate, and morphological and protein-level verification of EVs. Studies have shown that the function of EVs under physiological and pathological conditions may be affected by the external environment, including the composition and pH of the lysate. The common lysis reagent DTT can release EVs by disrupting the sputum protein polymer network, but smaller protein polymer fibers may subsequently co-isolate with the EVs, resulting in the presence of protein polymer fiber contaminants in the EV sample, which can reduce the purity of the EVs and affect the accuracy of subsequent proteomics analyses and other experimental results. Currently, differential centrifugation or PEG precipitation with a kit are commonly used to extract extracellular vesicles after sputum dissolution. Differential centrifugation is time-consuming (3-4 hours), consumes a lot of sample, causes significant vesicle damage, and results in a low recovery rate. PEG precipitation often introduces contamination from other proteins in the enzymatic digestion solution. Therefore, how to rapidly extract and enrich high-purity extracellular vesicles from sputum remains a difficult problem that needs to be solved urgently. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a method for extracting extracellular vesicles from induced sputum, which aims to solve the problems mentioned in the background technology.

[0006] The present invention provides a method for extracting extracellular vesicles from induced sputum, comprising the following steps:

[0007] Step S1: After collecting induced sputum by atomization, the sputum plug was picked out and dissolved in a pH 6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 to obtain an induced sputum solution rich in extracellular vesicles; the dissolution temperature was 37 ± 0.1°C, and the solution was digested on a shaker at a speed of 50 ± 1 rpm for 15 ± 1 min;

[0008] Step S2: Extracellular vesicles are extracted from the induced sputum solution by negative pressure size exclusion chromatography.

[0009] Furthermore, in step S1, after the induced sputum is collected by atomization, the sputum plug is picked out. The specific steps are: obtaining the patient's induced sputum sample by atomizing concentrated saline; weighing the centrifuge tube, picking out the sputum plug with tweezers, placing it into the centrifuge tube, and weighing it again to determine the mass of the sputum plug.

[0010] Furthermore, in step S1, a pH = 6.0 isotonic phosphate buffer solution containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 is added to the centrifuge tube; the centrifuge tube is then placed horizontally on a shaker for dissolution; after the dissolution is completed, the centrifuge tube is centrifuged to obtain a first supernatant, which is an induced sputum dissolution solution rich in extracellular vesicles from which cells are removed, wherein the centrifugation conditions are 3000 rpm, 4±0.1°C, and 5 min.

[0011] Furthermore, when the mass of the sputum plug is 2±0.5 g, the amount of pH=6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 added is 10 mL.

[0012] Furthermore, in step S2, the induced sputum solution is subjected to negative pressure size exclusion chromatography to extract extracellular vesicles, specifically the following steps:

[0013] Step S21: centrifuging the induced sputum solution to obtain a second supernatant, wherein the centrifugation conditions are 17000g, 4±0.1°C, 30±1min, and the second supernatant is the secondary supernatant;

[0014] Step S22: filtering the second supernatant through a 0.22 μm filter, and adding the obtained filtrate to a negative pressure size exclusion chromatography column;

[0015] Step S23: After the filtrate completely enters the negative pressure size exclusion chromatography column, phosphate buffer is added; the phosphate buffer is added in 6-8 times; the fractions generated after the first 1-2 additions and the last addition of phosphate buffer are drained, and the fractions generated after the other additions of phosphate buffer are all collected to obtain an extracellular vesicle suspension.

[0016] Furthermore, in step S22, the negative pressure size exclusion chromatography column is filled with 10 ml of agarose gel CL-6B.

[0017] Furthermore, in step S22, the constant negative pressure of the negative pressure size exclusion chromatography column is 1-100 μL / s.

[0018] Furthermore, in step S23, the amount of phosphate buffer added each time is 500 μL-1 mL, and the amount of extracellular vesicle suspension collected is 5 times the amount of phosphate buffer added each time.

[0019] Furthermore, 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. The obtained solid phase is a concentrated induced sputum extracellular vesicle suspension, and the centrifugation conditions are 4000g, 4±0.1°C, and 15±1min.

[0020] The present invention has the following beneficial effects:

[0021] (1) A multicomponent solubilizer of hydroxymethylcysteine ​​and Tween-20 is used to promote sputum dissolution and protect proteins and nucleic acids in extracellular vesicles from degradation; negative pressure size exclusion chromatography can accurately control the flow rate driven by negative pressure, ensuring the stable migration of extracellular vesicles in the chromatographic column, avoiding vesicle rupture caused by too fast flow rate or low separation efficiency caused by too slow flow rate.

[0022] (2) Compared with differential centrifugation, this method can be quickly implemented in clinical laboratories, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings:

[0024] Figure 1 This is a flow chart of the method for extracting extracellular vesicles from induced sputum according to Example 1 of the present invention.

[0025] Figure 2 The figure shows the number of extracellular vesicles and the electron micrograph of extracellular vesicles in the fractions collected by the negative pressure size exclusion chromatography column in Example 1 of the present invention; wherein:

[0026] Figure 2 A in the figure is the number of extracellular vesicle particles;

[0027] Figure 2 B is an electron micrograph of extracellular vesicles, scale bar: 100 nm.

[0028] Figure 3 Electron microscopic images of the extracellular vesicle suspensions of induced sputum in Example 1 of the present invention and Comparative Example 1, scale bar: 1.0 μm.

[0029] Figure 4 Particle size diagram of the induced sputum extracellular vesicle suspension in Example 1 of the present invention and Comparative Example 1; wherein:

[0030] Figure 4 A in FIG. 1 is a particle size distribution curve diagram of Example 1;

[0031] Figure 4 B in the figure is the particle size distribution curve of Comparative Example 1.

[0032] Figure 5 Immunoblotting identification of extracellular vesicle marker proteins of Example 1 of the present invention and Comparative Example 1.

[0033] Figure 6 This is a graph showing the extracellular vesicle extraction amount of Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is 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 intended to limit the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0036] An embodiment of the present invention provides a method for extracting extracellular vesicles from induced sputum, comprising the following steps:

[0037] Step S1: After collecting induced sputum by atomization, the sputum plug was picked out and dissolved in a pH 6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 to obtain an induced sputum solution rich in extracellular vesicles; the dissolution temperature was 37 ± 0.1°C, and the solution was digested on a shaker at a speed of 50 ± 1 rpm for 15 ± 1 min;

[0038] Step S2: Extracellular vesicles are extracted from the induced sputum solution by negative pressure size exclusion chromatography.

[0039] Specifically, in step S1, after the induced sputum is collected by atomization, the sputum plug is picked out. The specific steps are: obtaining the patient's induced sputum sample by atomizing concentrated saline; weighing the centrifuge tube, picking out the sputum plug with tweezers, placing it in the centrifuge tube, and weighing it again to determine the mass of the sputum plug.

[0040] Specifically, in step S1, a pH = 6.0 isotonic phosphate buffer solution containing 0.1% wt hydroxymethylcysteine ​​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 a first supernatant, which is an induced sputum dissolution solution rich in extracellular vesicles from which cells are removed, wherein the centrifugation conditions are 3000 rpm, 4±0.1°C, and 5 min.

[0041] Specifically, when the mass of the sputum plug is 2±0.5 g, the amount of pH=6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 added is 10 mL.

[0042] Specifically, in step S2, the induced sputum solution is subjected to negative pressure size exclusion chromatography to extract extracellular vesicles, and the specific steps are as follows:

[0043] Step S21: centrifuging the induced sputum solution to obtain a second supernatant, wherein the centrifugation conditions are 17000g, 4±0.1°C, 30±1min, and the second supernatant is the secondary supernatant;

[0044] Step S22: filtering the second supernatant through a 0.22 μm filter, and adding the obtained filtrate to a negative pressure size exclusion chromatography column;

[0045] Step S23: After the filtrate completely enters the negative pressure size exclusion chromatography column, phosphate buffer is added; the phosphate buffer is added in 6-8 times; the fractions generated after the first 1-2 additions and the last addition of phosphate buffer are drained, and the fractions generated after the other additions of phosphate buffer are all collected to obtain an extracellular vesicle suspension.

[0046] Specifically, in step S22, the negative pressure size exclusion chromatography column is filled with 10 ml of agarose gel CL-6B.

[0047] Specifically, in step S22, the constant negative pressure of the negative pressure size exclusion chromatography column is 1-100 μL / s.

[0048] Specifically, in step S23, the amount of phosphate buffer added each time is 500 μL-1 mL, and the amount of extracellular vesicle suspension collected is 5 times the amount of phosphate buffer added each time.

[0049] Specifically, 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. The obtained solid phase is a concentrated induced sputum extracellular vesicle suspension, and the centrifugation conditions are 4000g, 4±0.1°C, and 15±1min.

[0050] Reagents:

[0051] Multicomponent dissolving agent: 0.1 g hydroxymethylcysteine ​​and 1 mL Tween-20 are dissolved in 100 mL of pH 6.0 isotonic phosphate buffer and stored in aliquots at -80°C.

[0052] pH = 6.0 isotonic phosphate buffer: 8.01 g NaCl, 0.20 g KCl, 1.78 g Na2HPO4·2H2O, and 0.27 g KH2PO4, add 900 mL sterile double-distilled water, adjust the pH to 6.0 with hydrochloric acid, and make up to 1 L.

[0053] Device:

[0054] Negative pressure size exclusion chromatography column: The liquid outlet is connected to the collection bottle through a Luer connector. The collection bottle has a vent at the neck of the bottle, which can be connected to a vacuum pump. After the vent is connected to the pump, it can provide a constant negative pressure to the size exclusion chromatography column. After use, the negative pressure size exclusion chromatography column is cleaned by adding one times the column bed volume of phosphate buffer. After cleaning, the extracellular vesicle extraction of the next sample can be continued, or 20% ethanol can be added and stored in a 4°C refrigerator.

[0055] Example 1:

[0056] (1) After collecting induced sputum by atomization, 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 PH=6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 to the centrifuge tube. Place the 15 mL centrifuge tube horizontally in 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, the centrifugation temperature is 4°C, and the centrifugation time is 5 min. The obtained supernatant is the induced sputum solution with cells removed. Transfer the supernatant to a new 15 mL centrifuge tube.

[0057] (2) The induced sputum solution was centrifuged at 17,000 rpm, 4°C, and 30 min to obtain a secondary supernatant; the secondary supernatant was filtered through a 0.22 μm filter, and the obtained filtrate was added to a negative pressure size exclusion chromatography column, wherein the negative pressure size exclusion chromatography column was filled with 10 ml of agarose gel CL-6B, and the amount of the secondary supernatant was 1 ml; after the filtrate completely entered the negative pressure size exclusion chromatography column, phosphate buffered saline (PBS) was added in 8 times, and the amount of phosphate buffer added each time was 500 μl; the fractions generated after the first 1-2 times and the last time of adding phosphate buffer were emptied, and the fractions generated after the 3rd to 7th times of heating the phosphate buffer were collected to obtain 2.5 mL of extracellular vesicle suspension;

[0058] (3) Add 2.5 mL of extracellular vesicle suspension to an ultrafiltration centrifuge tube (100 KDa / 4 ml), centrifuge at 4000 g, 4 °C, and 5 min until the solid phase accounts for 10% of the total extracellular vesicle suspension. The obtained solid phase is 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.

[0059] The process of step (1), step (2) and step (3) in Example 1 is as follows: Figure 1 As shown, the whole process takes 1 to 2 hours.

[0060] Phosphate buffer was added in batches, and the extracellular vesicle particle number and extracellular vesicle electron microscopy results of the fractions collected by negative pressure size exclusion chromatography were as follows: Figure 2 As shown, the results showed that the 3rd to 7th fractions met the requirements. Therefore, in step (2), the fractions generated after heating the phosphate buffer for the 3rd to 7th times were collected to obtain 2.5 mL of extracellular vesicle suspension with a high recovery rate, which can be used for subsequent protein and RNA detection.

[0061] Comparative Example 1:

[0062] Refer to Example 1, except that 10 mL of pH=6.0 isotonic phosphate buffer containing 0.1%wt hydroxymethylcysteine ​​and 0.1%vol Tween-20 is replaced by 10 mL of 0.1% DTT.

[0063] Comparative Example 2:

[0064] Refer to Example 1, except that 10 mL of pH 6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 is replaced by 10 mL of 0.1% trypsin.

[0065] Result analysis:

[0066] The electron microscopic results of the induced sputum extracellular vesicle suspension in Example 1 and Comparative Example 1 are as follows: Figure 3 As shown; the particle size results of the induced sputum extracellular vesicle suspension in Example 1 and Comparative Example 1 are as shown Figure 4 As shown; the results of immunoblotting identification of extracellular vesicle marker proteins in Example 1 and Comparative Example 1 are as follows Figure 5 As shown; the extracellular vesicle extraction amount of Example 1 and Comparative Example 1 is as shown Figure 6 As shown, Figure 6 The dissolving solution 1 is a pH=6.0 isotonic phosphate buffer solution containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20, and the dissolving solution 2 is a 0.1% DTT solution.

[0067] The results showed that, under the premise that the sputum source and sampling weight and volume were the same, compared with 0.1% DTT dissolution, 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 in pH = 6.0 isotonic phosphate buffer solution could reduce the protein polymer fiber contaminants (such as Figure 3 As shown in Figure 2 ); Under the premise that the sputum source and sampling weight and volume are the same, compared with 0.1% DTT dissolution, 0.1%wt hydroxymethylcysteine ​​and 0.1%vol Tween-20 in pH=6.0 isotonic phosphate buffer can inhibit the rupture of extracellular vesicles and obtain more extracellular vesicles (such as Figure 4 and Figure 5As shown), that is, compared with Comparative Example 1, the extracellular vesicle marker protein is more enriched in the induced sputum extracellular vesicle suspension extracted in Example 1, and the extracellular vesicle suspension is of qualified quality at the protein level.

[0068] In summary, the present invention utilizes a multicomponent dissolving agent composed of hydroxymethylcysteine ​​and Tween-20 to promote sputum dissolution and protect proteins and nucleic acids in extracellular vesicles from degradation. Vacuum size exclusion chromatography precisely controls the flow rate driven by negative pressure, ensuring stable extracellular vesicle migration within the chromatographic column and avoiding vesicle rupture caused by excessive flow rates or inefficient separation due to excessively slow flow rates. Compared to differential centrifugation, this method can be rapidly implemented in clinical laboratories, requires less sample, has a high recovery rate, shortens the detection cycle (1-2 hours), and improves diagnostic and treatment efficiency. This method provides more efficient, simple, economical, and reliable technical support for biomedical research.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting extracellular vesicles from induced sputum, characterized in that: The following steps are involved: Step S1: After collecting induced sputum by atomization, the sputum plug was picked out and dissolved in a pH 6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 to obtain an induced sputum solution rich in extracellular vesicles; the dissolution temperature was 37 ± 0.1°C, and the solution was digested on a shaker at a speed of 50 ± 1 rpm for 15 ± 1 min; Step S2: Extracellular vesicles are extracted from the induced sputum solution by negative pressure size exclusion chromatography, specifically as follows: Step S21: centrifuging the induced sputum solution to obtain a second supernatant, wherein the centrifugation conditions are 17000g, 4±0.1°C, 30±1min, and the second supernatant is the secondary supernatant; Step S22: filtering the second supernatant through a 0.22 μm filter, and adding the obtained filtrate to a negative pressure size exclusion chromatography column; Step S23: After the filtrate has completely entered the negative pressure size exclusion chromatography column, phosphate buffer is added; The phosphate buffer is added in 6-8 times; the fractions generated after the first 1-2 times and the last time the phosphate buffer is added are drained, and the fractions generated after the other additions of the phosphate buffer are all collected to obtain an extracellular vesicle suspension.

2. The method for extracting extracellular vesicles from induced sputum according to claim 1, wherein: In step S1, after the induced sputum is collected by atomization, the sputum plug is picked out. The specific steps are: obtaining the patient's induced sputum sample by atomizing concentrated saline; weighing the centrifuge tube, picking out the sputum plug with tweezers, placing it in the centrifuge tube, and weighing it again to determine the mass of the sputum plug.

3. The method for extracting extracellular vesicles from induced sputum according to claim 2, wherein: In step S1, a pH = 6.0 isotonic phosphate buffer solution containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 is added to the centrifuge tube; the centrifuge tube is then placed horizontally on a shaker for dissolution; after dissolution is completed, the centrifuge tube is centrifuged to obtain a first supernatant, which is an induced sputum solution rich in extracellular vesicles from which cells are removed, wherein 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 plug is 2±0.5 g, the amount of pH=6.0 isotonic phosphate buffer containing 0.1% wt hydroxymethylcysteine ​​and 0.1% vol Tween-20 added is 10 mL.

5. The method for extracting extracellular vesicles from induced sputum according to claim 4, wherein: In step S22, the negative pressure size exclusion chromatography column is filled with 10 ml of agarose gel CL-6B.

6. The method for extracting extracellular vesicles from induced sputum according to claim 5, wherein: In step S22 , the constant negative pressure of the negative pressure size exclusion chromatography column is 1-100 μL / s.

7. The method for extracting extracellular vesicles from induced sputum according to claim 6, wherein: In step S23, the amount of phosphate buffer added each time is 500 μL-1 mL, and the amount of extracellular vesicle suspension collected is 5 times the amount of phosphate buffer added each time.

8. The method for extracting extracellular vesicles from induced sputum according to claim 7, wherein: 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. The obtained solid phase is a concentrated induced sputum extracellular vesicle suspension, wherein the centrifugation conditions are 4000g, 4±0.1°C, and 15±1min.

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