WO2017154951A1 - Method for recovering extracellular vesicles - Google Patents

Abstract

Provided is a method for recovering extracellular vesicles having excellent separation efficiency for separating and trapping the extracellular vesicles such as exosomes in a dispersion, the method furthermore making it possible to efficiently recover the trapped extracellular vesicles. A method for recovering extracellular vesicles in which a dispersion containing extracellular vesicles having an average particle size of 10-700 nm is brought into contact with a perforated substrate comprising a porous material, whereby extracellular vesicles from the dispersion are trapped on a perforated substrate and separated, and then the trapped extracellular vesicles are recovered from the perforated substrate.

Description

Method for collecting extracellular vesicles

The present invention relates to a method for collecting extracellular vesicles, in which the extracellular vesicles are separated from the dispersion liquid containing the extracellular vesicles and then the separated extracellular vesicles are collected.

In order to diagnose and evaluate the state of living organisms such as the presence and progression of disease, it has been widely used to use substances such as proteins contained in blood as biomarkers (bioindicator compounds). Specifically, using this biomarker, serum or urine is used as a sample, and ovarian cancer, prostate cancer, breast cancer, hepatocellular cancer, kidney cancer, pancreatic cancer, head and neck cancer, diabetes Evaluation of diseases such as myocardial infarction, alcoholism, renal damage caused by contrast media, bladder cancer, and coronary arteriosclerosis.

In conducting this evaluation, substances that can be biomarkers for the target disease must be obtained to the extent that they can be measured. For example, blood and urine contain various compounds, and it is difficult to measure them as they are. Therefore, it is possible to measure by increasing the concentration of the substance to be measured, such as separating the biomarker from such a sample, and performing a predetermined process for removing impurities that adversely affect the measurement.

Recently, as such a biomarker, a minute extracellular vesicle called an exosome is expected as a new biomarker and has been actively studied. These exosomes are contained in various body fluids of the living body, and in the body fluids, the average particle diameter is approximately 10 to 500 nm and exists as substantially spherical fine particles.

In order to measure such minute exosomes, ultracentrifugation, precipitation, filter, etc. (see, for example, Patent Document 1), antibody-bound magnetic bead method, chromatogram method, method using nanowire device, etc. ( For example, according to Patent Documents 2 to 4, a technique for separating exosomes in body fluids is known.

Separation by ultracentrifugation is the most widely performed, but it requires a very large centrifugal force, so that the exosome that is the separation target may be destroyed. In addition, the processing requires several tens of hours and the recovery rate is not high, so the processing efficiency is not so high. Furthermore, the equipment used is expensive and relatively large due to the special equipment capable of ultracentrifugation.

Also, according to the precipitation method, it is not necessary to apply a large centrifugal force and it can be easily isolated with a reagent, but it is necessary to leave it for several hours to overnight. In addition, the separation accuracy from impurities such as proteins is poor, and the purity of the obtained exosome is low, so the measurement accuracy does not increase.

In addition, according to the filter method, it is not necessary to apply a large centrifugal force, and a resin filter can be used for separation with a few minutes of filtration, and the treatment efficiency is relatively good. However, since its trapping efficiency is low, and it is highly likely that other impurities are trapped on the filter at the same time as the exosome, it is difficult to separate exosomes and impurities. Therefore, it is difficult to obtain exosomes of a predetermined purity or higher with high efficiency, and the measurement accuracy does not increase. In addition, since the exosomes captured by the filter cannot be recovered from the filter, the surface information of the exosome cannot be analyzed.

In addition, since the antigen-antibody magnetic bead method is based on an antigen-antibody reaction, the separation efficiency varies depending on the presence of exosome membrane protein, and stable measurement is difficult. In addition, in a sample containing a large amount of impurities such as protein (for example, blood), the reaction with the antibody is inhibited by the protein, and the reaction efficiency is lowered. Furthermore, since it is necessary to prepare and prepare magnetic beads on which a specific antibody is immobilized in advance, it takes time and cost. Since the separated exosomes are bound with magnetic beads, there are restrictions such as the need for post-processing in analysis, etc., or the use of analytical methods that do not cause the presence of magnetic beads. End up.

The chromatogram method is obtained by passing and eluting a sample in the chromatogram, and the apparatus is simple and the operation is simple. However, it is difficult to efficiently obtain high purity exosomes, and the processing efficiency is poor.

In the nanowire device method, nanowires are provided in the flow path of the specimen and exosomes are captured by adsorption, and the separation efficiency cannot be improved beyond a certain level. Moreover, since it is necessary to provide a special nanowire on a board | substrate, an effort and cost start.

Special table 2010-537158 JP 2013-128452 A International Publication No. 2014/168020 Japanese Patent No. 5291241

Therefore, the present invention provides an extracellular small cell that is excellent in separation efficiency and capable of recovering the captured extracellular vesicle with high purity when separating and capturing an extracellular vesicle such as an exosome contained in the dispersion. The purpose is to provide a method for collecting cells.

In the method for recovering extracellular vesicles of the present invention, a dispersion containing extracellular vesicles having an average particle size of 10 to 700 nm (preferably extracellular vesicles having an average particle size of 10 to 500 nm) is obtained by pores made of a porous material. The extracellular vesicles are collected from the perforated substrate after the extracellular vesicles are captured and separated from the dispersion by contacting the free substrate. To do.

According to the method for recovering extracellular vesicles of the present invention, the extracellular vesicles in the dispersion can be efficiently separated and captured, and the captured extracellular vesicles can be recovered with high purity. Therefore, extracellular vesicles that can be provided as they are for observation, inspection, etc. of extracellular vesicles can be obtained at low cost by a simple operation.

Hereinafter, a method for collecting extracellular vesicles according to an embodiment of the present invention will be described in detail.
As described above, the method for recovering extracellular vesicles of this embodiment is first a dispersion containing extracellular vesicles having an average particle size of 10 to 700 nm (preferably extracellular vesicles having an average particle size of 10 to 500 nm). Is brought into contact with a perforated substrate made of a porous material, whereby extracellular vesicles are captured from the dispersion and separated. Next, extracellular vesicles captured by the perforated substrate are separated from the perforated substrate and collected.

<Dispersion>
In describing the method for collecting extracellular vesicles of this embodiment in detail, first, a dispersion containing extracellular vesicles to be collected will be described.

The dispersion is not particularly limited as long as it is a dispersion containing extracellular vesicles dispersed in a liquid medium. Here, the liquid medium is not particularly limited as long as it is a liquid that can contain extracellular vesicles in a dispersed manner and has a viscosity that can pass through the pores of the porous substrate described later. .

As the dispersion liquid, a body fluid containing extracellular vesicles and a fluid obtained by treating the body fluid are usually used, and specifically, obtained by treating blood, lymph, tissue fluid, urine, sweat, or blood. Examples include plasma and serum.

Extracellular vesicles are sac-like minute structures wrapped in membranes contained in animal and plant cells, and function to store substances in cells and transport substances in and out of cells. . Specific examples of the extracellular vesicle include microvesicles, apoptotic bodies, exosomes, and the like. This extracellular vesicle is generally separated from the cytoplasmic matrix by a phospholipid bilayer, and the inside of the extracellular vesicle can be set to a predetermined condition, and may be used as various reaction fields.

That is, each of the extracellular vesicles has a specialized function, and the characteristics expressed to the outside and the types of substances contained therein are different depending on the type. Therefore, analysis of diseases and the like may be possible by analyzing extracellular vesicles themselves or substances contained in extracellular vesicles.

Here, the average particle size of the extracellular vesicles to be collected is 10 to 700 nm, preferably 10 to 500 nm, more preferably 10 to 300 nm, particularly preferably 10 to 200 nm, and further preferably 10 to 100 nm. . In this specification, the average particle diameter of the extracellular vesicle is a 50% integrated value (D ₅₀ ) obtained from a volume-based particle size distribution measured by a laser diffraction / scattering method, microscopic observation or the like.

<Perforated board>
The perforated substrate used here is a plate-like substrate capable of separating and capturing extracellular vesicles having an average particle diameter of 10 to 500 nm contained in the dispersion. This perforated substrate is composed of a porous body having a large number of pores in order to enable solid-liquid separation between extracellular vesicles and liquid. Examples of the material constituting the porous body include glass, resin, ceramics and the like.

Here, the average pore diameter of the pores of the perforated substrate is preferably 5 to 2500 nm, more preferably 5 to 1000 nm, still more preferably 5 to 550 nm, or 10 to 600 nm. Further, the average pore diameter is preferably 10 to 300 nm, more preferably 20 to 300 nm, still more preferably 20 to 200 nm, and most preferably 50 to 150 nm. Here, the average pore diameter refers to the peak top pore diameter determined based on the pore diameter distribution obtained by the BHJ method from the nitrogen adsorption isotherm by the gas adsorption method.

The average pore diameter is 30 to 800% of the average particle diameter of extracellular vesicles to be separated and trapped (that is, the ratio when the average particle diameter of extracellular vesicles is set as a reference (100%)). 30 to 800%) is preferable. More preferably, it is 30 to 500%, further preferably 30 to 200%, still more preferably 50 to 110%, and particularly preferably 60 to 100%. By setting the average pore diameter to 800% or less with respect to the average particle diameter of the extracellular vesicles, the extracellular vesicles can be reliably captured on the glass substrate, and the separation efficiency is improved. In addition, by making the average pore diameter larger than 30% of the average particle diameter of the extracellular vesicles, it is possible to suppress pressure loss because it does not have an excessively small pore diameter, making the extracellular vesicles efficient. Can be separated.

By setting the average pore size in such a range, a substance having an excessively small particle size (for example, albumin, IgG, transferrin and other blood proteins) while capturing the target extracellular vesicles with a porous substrate. Etc.) can pass through the perforated substrate together with the liquid medium of the dispersion liquid, and capture the target extracellular vesicles.

When the average pore diameter is 30% or more and 110% or less of the average particle diameter of the extracellular vesicle, the extracellular vesicle is mainly captured on the surface of the porous substrate. Therefore, it is preferable that extracellular vesicles are easily detached from the perforated substrate during the collection operation described later. In addition, since the extracellular vesicles captured at this time are captured at the same height on the surface of the perforated substrate, it can be efficiently observed even when the surface of the perforated substrate is directly observed with a microscope or the like.

Further, when this average pore size is more than 110% and less than 800%, particularly more than 110% and less than 500% of the average particle size of the extracellular vesicles, the extracellular vesicles captured in the pores of the porous substrate The number increases. Therefore, when performing the recovery operation after the separation operation, the extracellular vesicles hardly come out from the pores, and the recovery efficiency is slightly reduced. However, even when the average particle size of the extracellular vesicles is larger than the average particle size, it can be captured well by the perforated substrate. Since the pressure loss does not increase unnecessarily, the separation operation can be performed easily.

A porous substrate having an average pore diameter of more than 500% of the average particle diameter of extracellular vesicles further increases the number of extracellular vesicles trapped in the pores of the porous substrate. This sample is preferably used when handling a sample having a viscosity higher than that of water. On the other hand, a porous substrate having an average pore diameter of more than 500% of the average particle size of the extracellular vesicles has an average fine substrate thickness from the viewpoint of reliably capturing the extracellular vesicles in the pores. The pore size is preferably 100 times or more, more preferably 500 times or more.
In this embodiment, the recovery step is essential, but the perforated substrate in which the extracellular vesicles are trapped in the pores as described above can be used for diagnosis as it is. is there. For example, in order to analyze miRNA and the like present inside extracellular vesicles, it is possible to destroy the extracellular vesicle membrane captured inside the substrate and extract the internal information.

The perforated substrate used here is, on a volume basis, 90% or more of the pores in the perforated substrate are 60 to 140% of the peak top pore size in the pore size distribution (that is, the peak top pore size). In the range of 60 to 140%). This can be paraphrased to be within ± 40% when the peak top pore size is set as the standard (0%) (Note that the standard for calculating the ratio at this time is the peak top pore size as described above. Is). Since the distribution range of the pore size distribution is narrow, extracellular vesicles can be separated and captured stably and efficiently. Further, it is preferable that 95% or more of the pores are included in the range of 60 to 140% of the peak top pore size in the pore size distribution.

The porosity of the perforated substrate is preferably 20 to 90% by volume, more preferably 30 to 80% by volume, and particularly preferably 40 to 70% by volume from the viewpoint of not excessively reducing the strength. . In the present specification, the porosity is a value calculated from the apparent density and the true density of the glass substrate. The apparent density and the true density are obtained by the pycnometer method.

Further, since the perforated substrate is a porous body as described above, the conditions under which the solid-liquid separation of the dispersion liquid can be performed stably and reliably were examined. As a result, it is effective that the ratio of the area of the main surface to the plate thickness of the perforated substrate and the average pore diameter (area / plate thickness / Log ₁₀ (average pore diameter)) has a predetermined relationship. Found.

That is, in the perforated substrate of this embodiment, the ratio of the area of the principal surface to the plate thickness and the average pore diameter (area (cm ² ) / plate thickness (mm) / Log ₁₀ (average pore size (nm)) is 4 0.5 cm ² / mm or less, preferably 0.01 to 3.5 cm ² / mm, and more preferably 0.01 to 2.5 cm ² / mm. Preferably, it is within the range of 0.01 to 2 cm ² / mm, and by satisfying such a relationship, it is resistant to high pressure during separation and capture of extracellular vesicles in the dispersion. Furthermore, by satisfying such a relationship, even if the plate thickness is reduced to 0.5 mm or less, the occurrence of warping of the filter can be significantly suppressed. Good pressure loss during capture Arbitrariness.

The thickness of the perforated substrate is preferably 0.1 to 5 mm, and more preferably 0.1 to 1 mm. The area of the perforated substrate is preferably 0.01 to 10.5 cm ² , more preferably 0.01 to 6 cm ² , 0.1 to 6 cm ² , 0.2 to 6 cm ² , and 0.3 to 6 cm ^2. Is particularly preferred. The shape of the filter is not particularly limited as long as it is a plate shape. For example, the shape in plan view may be various shapes such as a polygon such as a triangle and a quadrangle, and a circle such as a perfect circle and an ellipse. In particular, when separating and capturing the extracellular vesicles in the dispersion, a circular shape, particularly a perfect circle, is preferable because the load applied from the liquid component becomes uniform and the strength is improved.

Further, the glass constituting the perforated substrate is not particularly limited as long as it has the above characteristics. Since it is easy to form the characteristic pore diameters described above, a phase separation glass in which spinodal phase separation is generated by heat treatment or the like is obtained by partially dissolving a soluble portion by acid treatment or the like. It is preferable to be a glass substrate.

Further, it is preferable that the perforated substrate has heat resistance and chemical resistance. Even when heat treatment is performed in the separation operation or the dispersion liquid to be used is acidic or alkaline, such physical and chemical durability is good, so that separation and capture operations can be performed stably. It can be carried out.

<Method for manufacturing perforated substrate>
As the perforated substrate of the present embodiment, a perforated glass substrate can be manufactured by, for example, a method of manufacturing via phase-separated glass or a method of forming mechanically uniform and fine through holes. The resin-made perforated substrate can be manufactured by a known resin filter manufacturing method such as a phase change method, a stretching method, or a radiation method. Hereinafter, a method for producing a glass perforated substrate via phase separation glass will be described as an example.

The production method via the phase-separated glass includes a phase separation heat treatment step in which a glass plate as a material is phase-separated by heat treatment, a soluble portion is dissolved by acid treatment or the like in the phase-separated glass plate, It can carry out by the porous-ized process made porous. Hereinafter, each step will be described.

First, the glass plate used as the material used here is not particularly limited as long as it is made of glass that undergoes phase separation by spinodal decomposition. Examples of such glass include silicon oxide-boron oxide-alkali metal oxide, silicon oxide-boron oxide-alkali metal oxide, at least one of alkaline earth metal oxide, zinc oxide, aluminum oxide, and zirconium oxide. Examples thereof include a glass containing a seed, a silicon oxide-phosphate-alkali metal oxide, a silicon oxide-boron oxide-calcium oxide-magnesium oxide-aluminum oxide-titanium oxide, and the like.

More specifically, SiO ₂ —B ₂ O ₃ —Na ₂ O, SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —Na ₂ O, SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ — CaO—MgO, SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —Na ₂ O—K ₂ O—CaO—MgO, SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —Li ₂ O—Na ₂ O—MgO, SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —Li ₂ O—Na ₂ O—CaO, SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —Na ₂ O—K ₂ O— CaO-ZrO _{2 system, SiO 2 -B 2 O 3 -Na} 2 O _{-based, SiO 2 -B 2 O 3 -CaO} -MgO-Al 2 O 3 -TiO 2 system include glass etc. is.

Among these, glass having a matrix composition of silicon oxide-boron oxide-alkali metal oxide is preferable, and the content of silicon oxide in the glass is preferably 45 to 80 mol%, more preferably 50 to 80 mol%, and 55 ˜80 mol% is more preferable, and 60 to 70 mol% is particularly preferable.

Glass that undergoes phase separation by spinodal decomposition is a glass having phase separation. For example, in the case of a borosilicate glass having silicon oxide-boron oxide-alkali metal oxide, the phase separation is performed by heat treatment to form a silicon oxide-rich phase and an alkali metal oxide-boron oxide-rich phase inside the glass. And phase separation.

Generally, glass can be phase-separated by heat-treating the glass as described above. Since the phase separation state formed in accordance with the heating temperature and the processing time changes in this heat treatment, the heat treatment may be set to a condition that obtains desired characteristics. The higher the heating temperature and the longer the treatment time, the more the phase separation state proceeds. As a result, a porous glass having a larger pore diameter can be obtained. In addition, changes in heating temperature have a large effect on the progress of phase separation, but changes in processing time have little effect on the progress of phase separation. Therefore, when obtaining a desired pore size, a rough range is defined by the heating temperature. It is better to perform precise control over the processing time. For example, the heating temperature is preferably in the range of 400 to 800 ° C., and the treatment is preferably performed in the range of 10 minutes to 200 hours, more preferably 10 minutes to 100 hours. This condition is particularly preferable in the borosilicate glass described above.

When manufacturing glass, those that are phase-separated at the melt stage at the time of melting the raw material include phase separation heat treatment, so the individual phase separation heat treatment as described above is performed. Can be omitted.

Next, the phase-separated glass (phase-separated glass) is subjected to an acid treatment to bring the alkali metal oxide-boron oxide rich phase, which is an acid-soluble component, into contact with an acid solution and dissolved and removed. The acid solution used here is not particularly limited as long as it can dissolve the soluble components, and examples thereof include organic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid, and acetic acid, and combinations thereof. Of these, inorganic acids such as hydrochloric acid and nitric acid are preferred.

Such an acid solution is preferably an aqueous solution, and the acid concentration may be appropriately set to an arbitrary pH. In this acid treatment, the temperature of the solution may be in the range of room temperature to 100 ° C., and the treatment time may be about 10 minutes to 200 hours, more preferably about 10 minutes to 150 hours. When the acid concentration is high or the temperature is high, the leaching process can be performed in a short time, but the frequency of glass cracking and warping due to leaching increases. In order to prevent this, an inorganic salt such as ammonium salt or borax may be added to the acid solution.

Furthermore, the acid-treated glass may be washed with at least one alkali solution and hot water. This washing treatment is performed for the purpose of dissolving and removing the residue generated by the acid treatment. At that time, the silicon oxide may be removed by hydrolysis or the like, and the porous formation may be promoted, and it can be used for adjusting the degree of the porous formation. In particular, the alkaline solution is effective for adjusting the degree of porosity, and hot water is effective for dissolving and removing the residue. Therefore, when both alkali solution treatment and hot water treatment are performed, it is preferable to perform the hot water treatment after the alkali solution treatment. Thus, when the hot water treatment is performed after the alkali solution treatment, the residue after the etching is effectively removed, and the transmittance of the glass substrate can be improved.

Examples of the alkali used here include alkaline solutions such as sodium hydroxide, potassium hydroxide, tetramethylammonium hydroxide, and ammonia, and an alkaline aqueous solution is preferable. In this alkali cleaning treatment, the alkali concentration of the alkali solution is in the range of 0.01 to 2.0 mol / L (0.01 to 2.0 N), particularly 0.1 to 2.0 mol / L (0. It may be set appropriately within the range of 1 to 2.0). In this alkali treatment, the temperature of the solution is preferably 10 to 60 ° C., and the treatment time is preferably 5 minutes to 10 hours, particularly 5 minutes to 2 hours.

Further, as the hot water, it is preferable to use pure water with few impurities, heated to 50 to 90 ° C., and the treatment time is 5 minutes to 2 hours.

Here, any one of the treatment with the alkaline solution and the treatment with hot water may be performed, but both may be performed. As described above, after the acid treatment, the acid-dissolved portion formed by phase separation by spinodal decomposition is dissolved by the acid treatment to form pores by washing with at least one of an alkaline solution and hot water. The holes are formed as continuous through-holes that are connected from one main surface to the other main surface with substantially the same hole diameter.

Depending on the treatment time of this acid treatment and the alkali and hot water treatment added thereto, the degree of vitrification of the glass body changes, and the size of the pores can be adjusted by performing each treatment for a long time. . Therefore, the processing conditions may be appropriately changed so that pores having a desired size can be obtained.

Also, the strength of the glass plate changes depending on the phase separation conditions and the treatment time such as acid treatment. The optimum phase separation condition depends on the glass composition, but in order to find the optimum phase separation condition, it is effective to examine, for example, a TTTT curve. The pore size can be reduced by advancing the phase separation in a temperature range lower by, for example, about 100 ° C. than the temperature range in which the phase separation is most likely to proceed, which is apparent from the TTT curve. In the dissolution treatment such as acid treatment, the strength tends to be lowered by increasing the treatment time. Therefore, treatment conditions such as phase separation conditions and acid treatment may be appropriately changed so that they can be used for separation of extracellular vesicles in a solution and ensure strength. That is, it can be adjusted by the composition of the glass plate, the phase separation heat treatment conditions (temperature and time), and the porosification conditions (liquid type, liquid composition, liquid concentration, treatment temperature, treatment time).

Further, in order to impart other physical and chemical functions to the perforated substrate, for example, after making it porous, a polymer or an inorganic film is formed on the surface by ALD, CVD, vacuum deposition, electron beam deposition, ion plating, By applying by supercritical film formation, dip coating, spin coating, spray coating, flow coating, squeegee coating, die coating, sputtering, ink jetting, etc., a predetermined functional layer is formed on the substrate surface, ultraviolet light, ozone, or Modification treatment may be performed by acid / alkali treatment or the like. As this functional layer or modification treatment, filtering performance is improved by hydrophilic treatment that improves the hydrophilicity of the surface, adsorption specificity for capturing only a specific substance is added, or a specific substance is adsorbed on the substrate surface. A glass substrate having desired characteristics can be obtained depending on the type of the functional layer to be formed and the modification treatment.

At this time, it is preferable to perform a cell non-adhesion treatment from the viewpoint of preventing the extracellular vesicles to be collected from adhering to the substrate and not peeling off. As this non-cell adhesion treatment, it is preferable to use a perforated substrate provided with a coating layer formed from a protein adhesion inhibitor. This coating layer may be formed by directly applying the protein adhesion preventing agent, or may be formed by dispersing the medium in a medium such as a solvent or a dispersion medium and then removing the medium. Here, a polymer having a structural unit having a biocompatible group can be used as the protein adhesion preventing agent. Specifically, in addition to a polymer having polyethylene glycol and 2-methacryloyloxyethyl phosphorylcholine as a structural unit, a fluoropolymer having a biocompatible group described in International Publication No. 2016/002796 is used. be able to.

Here, as the protein adhesion preventing agent, a polymer having a structural unit having a biocompatible group can be used. Specifically, in addition to a polymer having polyethylene glycol and 2-methacryloyloxyethyl phosphorylcholine as a structural unit, a fluorine-containing polymer having a biocompatible group described in International Publication No. 2016/002796 is used. be able to.

When a functional layer is formed by applying a protein adhesion inhibitor, the protein contained in blood, serum, etc. is prevented from adhering to the surface of the perforated substrate in the separation operation, and the protein is perforated together with the liquid medium. Easy to pass through the substrate. As a result, the substance captured by the perforated substrate is mainly extracellular vesicles, and the purity of the extracellular vesicles obtained by collection, which will be described later, can be increased.

Furthermore, when protein is adsorbed on the perforated substrate, extracellular vesicles may be fixed on the perforated substrate via the protein. However, when a coating layer formed of a protein adhesion inhibitor is used, In addition, it decreases, and in the collection operation described later, the extracellular vesicles are easily separated from the perforated substrate, and an effect of improving the recovery rate can be obtained. If the protein concentration is low, the extracellular vesicles that are charged and sticky like the protein may adhere directly to the substrate, but if a coating layer formed from a protein adhesion inhibitor is provided Similarly, adhesion of extracellular vesicles can also be prevented. Thereby, the improvement effect of a recovery rate is acquired.

And, when a coating layer formed from a protein adhesion inhibitor is provided, since the removal efficiency of the protein is improved as described above, noise in the test is also reduced, so that the collected extracellular vesicles are left as they are or Inspection and the like can be performed by simple processing, and the reliability of the inspection itself can be improved.

<Recovery operation of extracellular vesicles>
Next, a method for collecting extracellular vesicles to which the perforated substrate of this embodiment is applied will be described separately for each operation of the separation step and the collection step.

<Separation process>
This separation step is a step in which a dispersion containing extracellular vesicles is supplied onto the above-mentioned perforated substrate, which is a capturing means, and solid-liquid separation is performed. By this solid-liquid separation, the liquid medium constituting the dispersion liquid passes through the capturing means and is discharged from the lower part of the capturing means. On the other hand, extracellular vesicles which are solid components and are to be captured cannot pass through the pores of the porous substrate, but are captured by at least one of the surface and the pores. In addition, although it is a solid component, minute substances (for example, blood proteins such as albumin, IgG, and transferrin) pass through the perforated substrate together with the liquid medium and are separated from the extracellular vesicles.

In addition, as can be performed at this time the solid-liquid separation efficiently, it is preferable to adjust the viscosity of the dispersion before separation such that the 4 × 10 -3 ^{Pa ·} s or less, 2 × 10 ^-3 It is more preferable to set it to Pa · s or less. Examples of the liquid component for achieving such a viscosity include osmotic pressure adjusting solutions (such as physiological saline) using water, alcohol or the like as a solvent.

In addition, when supplying a dispersion liquid to the perforated substrate in this embodiment and making it pass, you may apply a pressure as needed and may let it flow. In applying the pressure, pressurization may be performed from the supplied dispersion side by using a pump or the like, or a centrifugal force may be applied by a centrifugal separator or the like. However, when the amount of the solution is very small, or when the viscosity of the solution is equal to or lower than that of water, it is possible to allow the solution to flow into the glass substrate only by permeation by capillary action.

When applying pressure in this trapping, the pressure is preferably 0.1 to 100 MPa. By setting the pressure to 0.1 MPa or more, solid-liquid separation can be promoted, and by setting the pressure to 100 MPa or less, breakage of the perforated substrate can be suppressed. The trapping means uses a cylindrical or tubular member made of glass, resin, rubber, or other material, and the dispersion is supplied to one of the perforated substrates by fixing the perforated substrate inside. It is preferable to form the dispersion liquid so that it can be filtered through a perforated substrate.

<Recovery process>
Next, the extracellular vesicles captured by the perforated substrate are collected.
In the collection, any method can be used without particular limitation as long as the captured extracellular vesicle is separated from the perforated substrate and collected. This recovery operation can be achieved by, for example, separating extracellular vesicles from the surface of the perforated substrate by washing, or moving and separating extracellular vesicles by a potential difference or a pressure difference.

For cleaning, the cleaning water is supplied to one side of the perforated board, the cleaning water is supplied from the side opposite to the filtration side of the perforated board, and the cleaning water is passed through the perforated board. It can be performed by a known cleaning method such as backwashing, dipping cleaning in which the perforated substrate is immersed in the stored cleaning water. Examples of the washing water used here include buffer solutions such as Tris buffer solution and phosphate buffer solution (PBS).

Also, in order to promote the peeling of the target object, it is effective to change the pH of the treatment liquid to near the isoelectric point of the target object or increase the salt concentration. For example, since exosomes are negatively charged in PBS around pH 7, exfoliation is facilitated by setting the pH to the acidic side of 4-6. Extreme fluidity, such as pH 1-2 or pH 13-14, can destroy exosomes. Further, peeling can be facilitated by setting the salt concentration to 0.1 to 1M. On the other hand, if the salt concentration is too high, it may also cause instability.

Further, in the above recovery, it is possible to easily remove the extracellular vesicles from the perforated substrate by applying ultrasonic vibration to the perforated substrate, applying an electric field, applying a pressure, or the like, thereby improving the recovery efficiency.

Here, the ultrasonic vibration is performed by applying vibration to a perforated substrate that has captured extracellular vesicles using a sound wave having a frequency of 10 kHz or more. At this time, the effect of applying ultrasonic energy is enhanced by adding a cleaning agent or other chemicals that reduce the surface tension of the cleaning water to reduce the surface tension of the cleaning water. On the other hand, in order to prevent destruction of extracellular vesicles to be collected, the frequency of the applied ultrasonic waves is preferably 100 kHz or less. The time for applying ultrasonic vibration is preferably about 1 to 30 minutes.

The electric field is applied by generating an electric field by applying a voltage across the perforated substrate. At this time, by determining the anode and the cathode according to the surface charge of the extracellular vesicle to be collected, it is possible to easily collect from the perforated substrate. For example, since an extracellular vesicle is easily charged with a negative charge in washing water, formation of an electric field induced at the anode is effective. In order to form this electric field, a voltage of 5 to 500 V is generally used, and 50 to 150 V is particularly preferable from the viewpoint of work efficiency and influence on cells. The electric field is applied at a low voltage for a long time or a high voltage for a short time depending on the purpose. For example, in the case of a low voltage, the voltage is 5 to 50 V for 10 to 60 minutes, and in the case of a high voltage, the voltage is 50. Examples of the treatment are 1 to 10 minutes at ~ 200V.

The pressure is applied by applying water pressure to the perforated substrate that has captured the extracellular vesicles by supplying cleaning water and collecting it in the flow direction of the cleaning water, or collecting the extracellular vesicles captured by centrifugation from the perforated substrate. A method of peeling off and collecting. The pressure varies depending on the trapping situation of extracellular vesicles. For example, when trapped near the surface of the perforated substrate, it can be recovered at a pressure of 0.01 MPa or more. Furthermore, in the case of a perforated substrate having a modified layer such as a non-adhesive treatment on the surface, it can be recovered at a pressure of 0.001 MPa or more. Further, when trapped inside the perforated substrate, it can be recovered at a pressure of 0.1 MPa or more. In addition, it is preferable to apply a pressure of 0.5 MPa or more from the viewpoint of the working efficiency of the recovery operation. Moreover, the upper limit of the applied pressure should just be a pressure which a perforated board | substrate can endure, for example, 100 MPa or less is preferable, 50 MPa or less is more preferable, and 10 MPa or less is further more preferable.

In the method for recovering extracellular vesicles of this embodiment, in order to separate and capture extracellular vesicles such as exosomes contained in the dispersion, the pore diameter and thickness of the porous substrate, the external force at the time of capturing, etc. By optimizing, it is possible to capture in a short time. Furthermore, by increasing the number of extracellular vesicles captured on the surface of the perforated substrate, recovery can be performed smoothly in a short time. In particular, when a coating layer formed of a protein adhesion inhibitor or a cell non-adhesive is provided on the surface of the perforated substrate, the working time can be further shortened and the collection can be performed efficiently.

In the above-described extracellular vesicle collection method, the example in which one perforated substrate is provided as the capturing means has been described. However, another perforated substrate having a different average pore diameter may be provided, or in the present embodiment. Although it does not correspond to the perforated substrate to be used, a glass filter or a resin filter having different average pore diameters may be provided. By combining filters with different pore sizes in this way, particles of different sizes contained in the dispersion can be separated and captured in stages to recover the target extracellular vesicles more efficiently. You can also.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these descriptions.

(Reference Example 1: Production of perforated substrate 1)
Three-component system of SiO ₂ 65%, B ₂ O ₃ 27%, Na ₂ O 8% in terms of oxide-based molar percentage, each raw material SiO ₂ , B ₂ O ₃ , Na ₂ CO ₃ particles The mixture was mixed so as to have a composition (SiO ₂ —B ₂ O ₃ —Na ₂ O) to obtain 900 g of a glass raw material.

This glass raw material was put in a platinum crucible, heated to 1500 ° C. with a resistance heating electric furnace, and melted. After defoaming and homogenization for 4 hours, the obtained molten glass was poured into a mold material, and cooled from the temperature of [glass transition point (Tg: 486 ° C.) + 50 ° C.] to room temperature at a rate of 30 ° C./min. A glass block was obtained.

The glass block was subjected to a phase separation treatment by heat treatment at 700 ° C. for 24 hours. The phase-separated glass block was cut and ground, and finally both surfaces were processed into mirror surfaces to obtain a plate-like glass having a diameter of 7 mmφ and a thickness of 1.0 mm.

Next, this plate-like glass was immersed in 1N HNO ₃ for 16 hours and leached, and then washed with 1N NaOH and 60 ° C. hot water to obtain a perforated substrate 1 (thickness 1.0 mm, average A pore diameter of 290 nm) was obtained.

In addition to the filter type and phase separation treatment conditions (heat treatment temperature, heat treatment time) of the obtained glass substrate, average pore diameter, distribution width in pore diameter distribution, plate thickness, main surface area, (area / plate thickness / Log ₁₀ (average pore size (nm)) were investigated and are shown together in Table 1. Here, the pore size distribution width was 0% at the peak top.

(Reference Example 2: Production of perforated substrate 2)
Using the same glass raw material as in Reference Example 1, a glass block was obtained under the same conditions.
The glass block was subjected to a phase separation treatment by heat treatment at 700 ° C. for 3 hours. The phase-separated glass block was cut and ground, and finally both surfaces were processed into mirror surfaces to obtain a plate-like glass having a diameter of 7 mmφ and a thickness of 1.0 mm.

Next, the plate glass was immersed in 1N HNO ₃ for 16 hours and leached, and then washed with 1N NaOH and 60 ° C. hot water to obtain a perforated substrate 2 (thickness 1.0 mm, average A pore diameter of 160 nm) was obtained.

In addition to the filter type and phase separation treatment conditions (heat treatment temperature, heat treatment time) of the obtained glass substrate, average pore diameter, distribution width in pore diameter distribution, plate thickness, main surface area, (area / plate thickness / Log ₁₀ (average pore size (nm)) were investigated and are shown together in Table 1. Here, the pore size distribution width was 0% at the peak top.

[Average pore size, pore size distribution]
The pore size distribution was determined by the BHJ method from the nitrogen adsorption isotherm by the gas adsorption method. The average pore diameter refers to the peak top pore diameter determined based on the obtained pore diameter distribution. The distribution width of the pore size distribution was expressed based on the peak top pore size (100%).

(Examples 1-2)
Dispersion solutions were prepared in which fluorescent beads having an average particle diameter of 50 nm, 100 nm, and 350 nm were dispersed in water. Next, the perforated substrates obtained in Reference Examples 1 and 2 were each fixed in a cylindrical tube so that the inside could be sealed, and a capturing means capable of processing 500 μL of the dispersion solution was produced.

Each of the obtained capturing means contains 500 μL of the prepared dispersion solution, centrifuged at 10,000 G (pressure 1.3 MPa) for 10 minutes with a centrifuge, and fluorescent beads in the dispersion solution with a perforated substrate. Captured. At this time, the capture rate of the fluorescent beads was calculated as follows by measuring the fluorescence intensity of the dispersion before and after filtration with a plate reader.
“Capture rate (%) = [(fluorescence intensity before filtration−fluorescence intensity after filtration) / fluorescence intensity before filtration] × 100”
That is, when the fluorescence intensity of the dispersion before filtration is 100%, the relative ratio of the fluorescence intensity of the filtrate after filtration is calculated, and the value obtained by subtracting it is the capture rate. The results are shown in Table 2.

Next, the perforated substrate on which the beads were captured was immersed in phosphate buffered saline (PBS), subjected to ultrasonic treatment for 15 minutes, and the beads were separated from the perforated substrate and collected. The fluorescence intensity in PBS was measured with a plate reader, and the recovery rate of each fluorescent bead was calculated. The results are shown in Table 3.

The above-described fluorescent bead capturing operation was performed again, and the perforated substrate was taken out from the capturing means without performing the recovery operation, and the perforated substrate surface was observed with a fluorescence microscope. At this time, the fluorescent beads captured on all the perforated substrates could be confirmed, and the main locations where they were captured were also confirmed. The results are also shown in Table 2. The place where the fluorescent beads are captured is the “surface” when the fluorescent beads are not captured to a depth greater than the particle diameter (partially exposed from the surface of the perforated substrate). Evaluation was made as “inside” when trapped at a depth equal to or greater than the diameter (the fluorescent beads were completely buried in the perforated substrate).

(Example 3)
A dispersion solution was prepared by dispersing HepG2-derived exosomes and proteins (bovine serum albumin) labeled with a PKH26GL cell linker kit (manufactured by SIGMA-ALDRICH, trade name) in a phosphate buffer solution (PBS). Here, the average particle size of HepG2-derived exosome is 127 nm.
The dispersion solution was subjected to exosome and protein capture tests using the perforated substrate obtained in Reference Example 1 in the same manner as in Example 1. The results are shown in Table 4. The exosome capture rate was calculated as follows by measuring the fluorescence intensity of the dispersion before and after filtration with a plate reader.
“Capture rate (%) = [(fluorescence intensity before filtration−fluorescence intensity after filtration) / fluorescence intensity before filtration] × 100”
That is, when the fluorescence intensity of the dispersion before filtration is 100%, the relative ratio of the fluorescence intensity of the filtrate after filtration is calculated, and the value obtained by subtracting it is the capture rate.
The protein capture rate was calculated in the same manner as described above from the protein concentration calculated from the absorbance of the dispersion before and after filtration using a spectrophotometer. That is, “capture rate (%) = [(absorbance before filtration−absorbance after filtration) / absorbance before filtration] × 100” was obtained.

(Reference Example 3: Production of perforated substrate 3)
[Synthesis of protein adhesion inhibitor]
0.886 g (3.0 mmol) of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 0.995 g (7.0 mmol) of butyl methacrylate (BMA) were weighed into a 300 mL three-necked flask and used as a polymerization initiator. 0.019 g of azobisisobutyronitrile (AIBN) and 9.50 g of ethanol (EtOH) as a polymerization solvent were added. The charge molar ratio of BMA and MPC was BMA / MPC = 70/30, the total concentration of monomers in the reaction solution was 20% by mass, and the initiator concentration was 1% by mass.
The flask was sufficiently purged with argon and sealed, and the polymerization reaction was carried out by heating to 75 ° C. for 16 hours. The reaction solution was ice-cooled and then added dropwise to diethyl ether to precipitate a polymer. The obtained polymer was sufficiently washed with diethyl ether and then dried under reduced pressure to obtain a white powdery polymer (A-1).
When the copolymer composition of the obtained polymer (A-1) was measured by ¹ H-NMR, it was BMA unit / MPC unit = 70/30 (molar ratio).

[Preparation of a perforated substrate having a layer formed from a protein adhesion inhibitor]
The polymer (A-1) was dissolved in ethanol so that its concentration was 0.05% by mass, and the perforated substrate 2 was immersed therein. After 30 minutes, it was taken out, blow-dried, and then vacuum-dried at room temperature for 1 hour to obtain a perforated substrate 3 having a coating layer of the polymer (A-1).

(Examples 4-5)
<Protein non-adsorption test>
A protein non-adsorption test was performed on the obtained perforated substrate 3 and the perforated substrate 2 that was not subjected to surface treatment as follows.
(1) Preparation of color developing solution and protein solution The color developing solution includes peroxidase color developing solution (3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), manufactured by KPL) 50 mL and TMB Peroxidase Substrate (manufactured by KPL). A mixture of 50 mL was used.
As the protein solution, a protein (POD-goat anti mouse IgG, manufactured by Biorad) diluted 16,000 times with a phosphate buffer solution (D-PBS, manufactured by Sigma) was used.
(2) Protein adsorption One perforated substrate 2 that has not been surface-treated and one perforated substrate 3 that has been surface-treated with the polymer (A-1) on a 24-well microplate into which 2 mL of the protein solution has been dispensed. It was immersed in another well and left at room temperature for 1 hour.
(3) Substrate cleaning Next, each substrate was washed four times with 4 mL of a phosphate buffer solution (D-PBS, manufactured by Sigma) containing 0.05% by mass of a surfactant (Tween 20, manufactured by Wako Pure Chemical Industries, Ltd.). Washed.
(4) Coloring solution dispensing Next, each perforated substrate after washing was immersed in a 24-well microplate into which 2 mL of the coloring solution had been dispensed, and a coloring reaction was performed for 7 minutes. The color reaction was stopped by adding 1 mL of 2N sulfuric acid.
(5) Preparation for absorbance measurement Next, 150 μL of liquid was taken from each well of the 24-well microplate and transferred to a 96-well microplate.
(6) Absorbance measurement and protein adsorption rate Absorbance was measured at 450 nm using MTP-810Lab (manufactured by Corona Electric Co., Ltd.), and the relative protein adsorption rate was evaluated by comparing the respective absorbances. At this time, the relative ratio when the absorbance without surface treatment was 1 was defined as the protein adsorption rate.
Table 5 shows the evaluation results of the obtained protein non-adsorbability. As a result, it was found that the amount of protein adsorbed on the substrate can be greatly reduced as compared with the case where the surface treatment was not performed. Therefore, when the collection operation is performed using the perforated substrate 3, the amount of mixed protein is small and extracellular vesicles can be collected with high purity. In addition, when collecting the extracellular vesicles that are sticky, they can be easily removed from the substrate, so that they can be efficiently recovered.

As described above, according to the method for recovering extracellular vesicles of the present embodiment, extracellular vesicles having an average particle size of 10 to 500 nm can be reliably captured and efficiently recovered. At this time, in the solid-liquid separation of the extracellular vesicles, it is required not to unnecessarily increase the pressure loss in order to pass the liquid, but the method of recovering the extracellular vesicles of this embodiment has this characteristic. Was also found to be good.

Also, in solid-liquid separation, it is possible to prevent extracellular vesicles from adhering to the surface of the perforated substrate by preventing the proteins contained in the body fluid from adhering to the surface of the perforated substrate serving as a filter. As a result, extracellular vesicles can be easily detached from the perforated substrate during collection, and the collection efficiency can be improved.

The method for recovering extracellular vesicles of this embodiment can recover extracellular vesicles efficiently and with good purity, and the recovered extracellular vesicles can be used as they are or as samples for inspection or the like by simple processing. In this collection, since extracellular vesicles are once captured on the perforated substrate, the extracellular vesicles can be observed on the substrate before the collection. Furthermore, this method of recovering extracellular vesicles can also be applied to capturing substances having a particle size larger than that of extracellular vesicles.

Claims (10)

1. By contacting a dispersion containing extracellular vesicles having an average particle size of 10 to 700 nm with a porous substrate made of a porous material, the extracellular vesicles are captured and separated from the dispersion by the porous substrate. After
   A method for recovering extracellular vesicles, wherein the captured extracellular vesicles are recovered from the perforated substrate.
2. The method for recovering extracellular vesicles according to claim 1, wherein the extracellular vesicles are extracellular vesicles having an average particle diameter of 10 to 500 nm.
3. The extracellular small particle according to claim 1 or 2, wherein an average pore diameter of pores of the porous substrate is in a range of 30% to 800% of an average particle diameter of the extracellular vesicle to be captured. How to recover cells.
4. The extracellular small particle according to claim 1 or 2, wherein an average pore diameter of pores of the porous substrate is in a range of 30% to 500% of an average particle diameter of the extracellular vesicle to be captured. How to recover cells.
5. 5. The method for recovering extracellular vesicles according to claim 3 or 4, wherein 90% or more of the pores are contained in a range of 60 to 140% of the peak top pore size in the pore size distribution on a volume basis.
6. The method for recovering extracellular vesicles according to any one of claims 1 to 5, wherein the perforated substrate is made of glass.
7. The method for recovering extracellular vesicles according to any one of claims 1 to 6, wherein the perforated substrate has a layer formed on the surface thereof from a protein adhesion inhibitor.
8. The method for recovering extracellular vesicles according to any one of claims 1 to 7, wherein the perforated substrate is subjected to a hydrophilic treatment on the surface thereof.
9. The dispersion according to any one of claims 1 to 8, wherein the dispersion liquid is blood or serum containing exosomes as the extracellular vesicles, and the exosomes are separated and recovered from proteins contained in the blood or serum. A method for collecting extracellular vesicles.
10. The method for recovering extracellular vesicles according to any one of claims 1 to 9, wherein at the time of the recovery of the extracellular vesicles, at least one treatment of applying an ultrasonic vibration, applying an electric field, and applying a pressure is performed.