WO2019150955A1

WO2019150955A1 - Filter member for capturing extracellular microparticles, kit for capturing extracellular microparticles and method for capturing extracellular microparticles

Info

Publication number: WO2019150955A1
Application number: PCT/JP2019/001131
Authority: WO
Inventors: 秀司山崎; 木原　直人; 馬場　嘉信; 博湯川; 大介小野島; 啓太青木
Original assignee: Ａｇｃ株式会社; 国立大学法人名古屋大学
Priority date: 2018-02-01
Filing date: 2019-01-16
Publication date: 2019-08-08
Also published as: JP2021052591A

Abstract

Provided are: a filter member for capturing extracellular microparticles, said filter member having a simple structure but yet being capable of achieving a high efficiency of capturing extracellular microparticles; a kit for capturing extracellular microparticles using the filter member; and a method for capturing extracellular microparticles using the filter member. The filter member 10 for capturing extracellular microparticles comprises: a plate-shaped filter 11 which is formed of a porous body capable of capturing extracellular microparticles and provided with a first principal surface 11a serving as a filter surface and a second principal surface 11b opposed to the first principal surface 11a; and an elastic member 12 which is disposed in the upper peripheral part of the first principal surface 11a and/or the lower peripheral part of the second principal surface 11b in the state of being elastically contacted with the filter 11.

Description

Extracellular particle capturing filter member, extracellular particle capturing kit, and extracellular particle capturing method

The present invention relates to an extracellular particle capturing filter member capable of separating and capturing extracellular particles, an extracellular particle capturing kit, and an extracellular particle capturing method.

Conventionally, filtration by a filter is well known as a technique for separating a solid substance contained in a solution from a liquid. Filters used for filtration are known to have various pore sizes depending on the purpose, and filters that can separate and capture very small particles (for example, extracellular fine particles) having an average particle size of 500 nm or less. Conventionally, a fiber type is well known (see, for example, Patent Document 1).

Further, as such a filter for capturing small particles, a filter using porous glass capable of capturing extracellular particles is also known (for example, see Patent Document 2).

Also known are filters for DNA separation using monolithic silica that can capture DNA as very small particles (see, for example, Patent Documents 3 and 4).

International Publication No. 2009-150834 International Publication No. 2016-136978 International Publication No. 2005-78088 Japanese Unexamined Patent Publication No. 2006-296220

By the way, in the fiber type filter described in Patent Document 1 or the like or the filter having a pore size of μm order, the pores are large, and therefore, when filtering a solution containing extracellular fine particles, resistance when passing through the filter is low. Small and the differential pressure applied to the filter is small.

On the other hand, in order to improve the capture efficiency of extracellular fine particles, it is considered that one method is to reduce the pore size of the filter to the order of nm, but at that time, the differential pressure during filtration increases. In addition, the present inventors have also found that the trapping efficiency of extracellular microparticles does not improve as expected under such conditions.

As a result of diligent investigations on the cause of this, the inventors of the present invention have found that when the pore size of the filter is reduced, the differential pressure increases as described above during filtration, so that the differential pressure is released between the filter and the container. It was found that a gap was formed, and the solution containing extracellular fine particles to be captured would flow out without being filtered from the gap.

Therefore, the present invention provides an extracellular particle capturing filter member capable of improving the capture efficiency of extracellular particles while having a simple configuration even during filtration using a filter having a small pore size, and the extracellular particle capturing It is an object of the present invention to provide an extracellular microparticle capturing kit and an extracellular microparticle capturing method using the filter member.

The filter member for capturing extracellular particulates of the present invention is made of a porous body capable of capturing extracellular particulates, and has a plate-like shape having a first principal surface serving as a filtration surface and a second principal surface facing the first principal surface. And an elastic member provided in elastic contact with the filter at the upper peripheral edge of the first main surface and / or the lower peripheral edge of the second main surface.

The kit for capturing extracellular microparticles of the present invention is a plate-like body composed of a porous body capable of capturing extracellular microparticles, and having a first main surface serving as a filtration surface and a second main surface facing the first main surface. An extracellular particle capturing filter member comprising: a filter; and an elastic member provided in elastic contact with the filter at an upper peripheral edge of the first main surface and / or a lower peripheral edge of the second main surface; And a container for holding the filter member.

In the extracellular particle capturing method of the present invention, the extracellular particle-containing solution containing extracellular particles is brought into contact with the extracellular particle capturing kit of the present invention, and the extracellular particle-containing solution is filtered using external force. The extracellular particles are captured by the filter member of the extracellular particle capturing kit.

According to the filter member for capturing extracellular particulates, the kit for capturing extracellular particulates, and the method for capturing extracellular particulates according to the present invention, a gap is formed between the filter and the container even though it has a simple configuration, and the target to be captured from the gap. It is possible to perform stable filtration with good trapping efficiency of the extracellular particles without causing a situation that the solution containing the extracellular particles is flowing out without passing through the filter. .

It is the side view which showed schematic structure of the filter member for extracellular particle capture | acquisition of this embodiment. 1B is a plan view of the filter member for capturing extracellular particles in FIG. 1A. FIG. FIG. 1B is a side sectional view of the extracellular particle capturing filter member of FIG. 1A. It is the sectional side view which showed schematic structure of the filter member for other extracellular particle capture | acquisition of this embodiment. It is the sectional side view which showed schematic structure of the filter member for another extracellular particle capture | acquisition of this embodiment. It is the sectional side view which showed schematic structure of the filter member for another extracellular particle capture | acquisition of this embodiment. It is the sectional side view which showed schematic structure of the filter member for another extracellular particle capture | acquisition of this embodiment. It is the sectional side view which showed schematic structure of the filter member for another extracellular particle capture | acquisition of this embodiment. It is the sectional side view which showed schematic structure of the kit for extracellular microparticle capture | acquisition of this embodiment.

Hereinafter, the filter member for capturing extracellular particles, the kit for capturing extracellular particles, and the method for capturing extracellular particles according to the present invention will be described in detail with reference to exemplary embodiments.

[Filter member for capturing extracellular particles]
As shown in FIGS. 1A to 1C, the filter member for capturing extracellular particulates according to the present embodiment has a plate-like filter 11 made of a porous body capable of capturing extracellular particulates, and elastically contacts the filter 11. And a filter member 10 for capturing extracellular fine particles. 1A to 1C are views showing a side view (FIG. 1A), a plan view (FIG. 1B), and a side sectional view (FIG. 1C) of the filter member 10 for capturing extracellular particles.

(filter)
The filter 11 used here is a plate-like filter having a first main surface 11a serving as a filtration surface and a second main surface 11b opposed to the first main surface. As described above, the filter 11 is made of a porous body capable of capturing extracellular fine particles, and an extracellular fine particle solution containing extracellular fine particles is supplied onto the first main surface 11a. Extracellular fine particles having an average particle size of 10 to 500 nm contained therein can be separated and captured, and the extracellular fine particles and the liquid medium can be solid-liquid separated.

The filter 11 is composed of a porous body having a large number of pores, and examples of the porous body include porous membranes. In addition, if classified by material, inorganic porous bodies such as monolithic silica and porous glass Examples thereof include organic porous bodies. In this porous body, a large number of holes are provided in a communication hole structure having a three-dimensional network structure (monolith structure).

The extracellular microparticles to be captured in this embodiment are nano to micro-sized microparticles that exist between living cells. These extracellular microparticles are derived from living bodies such as microvesicles and exosomes that are extracellular vesicles (endogenous), and those taken into the body from outside the body such as PM2.5, pollen, and nanoparticles (exogenous). )are categorized. The average particle size of the extracellular fine particles is 10 nm to 5 μm. In this specification, the average particle diameter of the extracellular fine particles is a 50% integrated value (D ₅₀ ) obtained from a volume-based particle size distribution measured by a laser diffraction / scattering method or microscopic observation, In the cumulative curve with 100%, the particle diameter is the point where the cumulative curve is 50%.

Examples of the extracellular microparticles include exogenous microparticles such as asbestos, carbon black, ink, colloidal particles, and viruses, and endogenous microparticles such as albumin, antibodies, and exosomes. In particular, extracellular vesicles such as exosomes It is attracting attention, and it is expected to be applied to drug discovery and diagnosis. Such extracellular vesicles have an average particle size of about 10 to 500 nm and an average particle size of about 50 to 200 nm.

An embodiment using a filter suitable for capturing such extracellular vesicles will be described below.
Here, the average pore diameter of the pores of the filter 11 is preferably 5 to 2500 nm, more preferably 10 to 1500 nm, still more preferably 10 to 1000 nm, still more preferably 40 to 1000 nm, and particularly preferably 50 to 800 nm. . Here, the average pore diameter refers to the peak top pore diameter determined based on the pore diameter distribution obtained from the nitrogen adsorption isotherm by the gas adsorption method by the BJH method or by the mercury intrusion method using a mercury porosimeter.

The average pore diameter is 30 to 800% of the average particle diameter of the extracellular fine particles to be separated and captured (that is, the ratio is 30 to 80% based on the average particle diameter of the extracellular fine particles (100%)). 800%) is preferable, more preferably 50 to 700%, and still more preferably 60 to 500%.

By setting the average pore size to 800% or less (8 times or less the average particle size) with respect to the average particle size of the extracellular fine particles, the extracellular fine particles can be captured on the filter 11 or in the pores.
In addition, by setting the average pore diameter to be larger than 30% of the average particle diameter of the extracellular fine particles, it is possible to prevent an excessively small pore diameter, thereby suppressing an increase in pressure loss and making the extracellular fine particles efficient. Can be separated. In addition, it is possible to pass a substance having an average particle diameter that is excessively smaller than the target extracellular fine particles and to capture only the target extracellular fine particles.

Further, by setting the average pore size to 200% or less (2.0 times or less of the average particle size) with respect to the average particle size of the extracellular fine particles, the extracellular fine particles can be captured mainly on the surface of the filter 11. Therefore, it is suitable for observing captured extracellular particles.

Further, when the average pore diameter is set to be more than 200% and not more than 800% of the average particle diameter of the extracellular particles, the number of extracellular particles captured in the pores of the filter 11 increases. Therefore, when the objective is observation as described above, it may be difficult to find target extracellular particles. However, even when the average particle size of the extracellular particles is larger than the average particle size, the filter 11 can capture the sample well. In this case, the pressure loss during solid-liquid separation Does not increase unnecessarily, so that the separation operation can be performed easily.

In the filter having an average pore diameter exceeding 300% of the average particle diameter of the extracellular fine particles, the number of extracellular vesicles captured in the pores of the filter 11 is further increased. It is preferably used when handling one having a higher viscosity than water. On the other hand, in the filter having an average pore diameter exceeding 300% of the average particle diameter of the extracellular fine particles, the thickness of the filter 11 is 100 times the average pore diameter from the viewpoint of reliably capturing the extracellular vesicles in the pores. Preferably, it is more than 500 times. On the other hand, from the viewpoint of preventing liquid permeability and sample loss, the thickness of the filter 11 is preferably 10,000 times or less of the average pore diameter.

In addition, the filter 11 in which the extracellular vesicles are trapped in the pores as described above can be used for diagnosis as it is. For example, in order to analyze RNA, DNA, protein, etc. existing inside and outside the extracellular vesicle, it is possible to destroy the membrane of the extracellular vesicle captured inside the substrate and extract the internal information. Some of the extracellular vesicle extracts used at this time have high viscosity, but by setting the average pore diameter to more than 200% of the average particle diameter of the extracellular microparticles, it is easy to pass through the filter 11. It is preferable.

Methods for measuring and analyzing the information obtained include microarray, next-generation sequencing (NGS), electrophoresis, polymerase chain reaction (PCR), mass spectrometry, etc. In any case, high capture rate of extracellular vesicles It is effective for analysis because a high-concentration extract can be obtained.

In addition, when using a sample with a low concentration of target extracellular vesicles, such as cell supernatant, repeatedly passing the sample through this filter allows the previously captured extracellular vesicles to enter the pores. It is possible to capture extracellular vesicles in the additional sample while capturing them, and to increase the amount of extracellular vesicles captured in the pores. Thereby, it is possible to concentrate extracellular vesicles by repeatedly inputting the amount even in a low concentration sample.

On a volume basis, 90% or more of the pores in the filter 11 are 60 to 140% of the pore diameter of the peak top in the pore diameter distribution (that is, the ratio when the peak top pore diameter is the reference (100%) is 60%. In the range of ~ 140%). This can be paraphrased to be within ± 40% when the peak top pore diameter is the standard (0%) (Note that the standard for calculating the ratio at this time is the peak top pore diameter as described above. Is). Due to the narrow distribution width of the pore size distribution, extracellular particles can be separated and captured stably and efficiently. Furthermore, it is more 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. Further, it is more preferable that 90% or more of the pores are included in the range of 70 to 130% of the peak top pore size in the pore size distribution.

The porosity of the filter 11 is preferably 20 to 95% by volume, more preferably 30 to 90% by volume, and still more preferably 40 to 85% by volume from the viewpoint of not excessively reducing the strength. Even more preferably, it is 45 to 70% by volume. 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.

In addition, the filter 11 has a ratio of the area of the main surface to the plate thickness of the filter 11 and the average pore diameter as a condition that the solid-liquid separation can be performed stably and reliably in the glass substrate having the specific pores ( The area / plate thickness / Log ₁₀ (average pore diameter)) is preferably a predetermined relationship.

That is, in the filter 11 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. 5 cm ² / mm or less is preferable, 3.5 cm ² / mm or less is more preferable, 0.01 to 3.0 cm ² / mm is more preferable, and 0.01 to 2 is preferable. even more preferably in the .5cm range of ² / mm, particularly preferably in the range of 0.1 ~ ^{2cm 2} / mm. by satisfying such a relationship, a solution containing the extracellular particulate Resistant to high pressures during separation and capture of extracellular particles in the filter, and by satisfying this relationship, warping of the filter occurs even when the plate thickness is reduced to 0.5 mm or less. Significant It can be suppressed. Moreover, by reducing the thickness, separation, preferably can reduce the pressure loss at the time of capture.

The thickness of the filter 11 is preferably 0.1 to 5 mm, more preferably 0.1 to 3 mm, and further preferably 0.3 to 2 mm. The area of the filter 11 is preferably 0.01 ~ 10.5cm ^2, more preferably 0.01 ~ 8 cm ^2, more preferably 0.01 ~ 6 cm ^2, even more preferably 0.05 ~ 3 cm ^2. 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. Among these, in the separation and capture of the extracellular fine particles in the extracellular fine particle-containing solution, a circular shape is preferable, and a perfect circle is more preferable because the load applied from the liquid component is uniform and the strength is improved. When the filter has a substantially disc shape, the diameter of the filter is preferably 1 to 30 mm.

Further, the bending strength of the filter 11 is preferably 20 MPa or more, and more preferably 100 MPa or more. When the bending strength is 20 MPa or more, it is possible to stably capture extracellular fine particles. This bending strength can be determined by a four-point bending test according to JIS R1601.

The glass constituting the filter 11 is not particularly limited as long as it has the above characteristics. In addition, since it is easy to form the above characteristic pore diameter, the glass constituting the filter 11 is soluble in a phase separation glass in which spinodal phase separation is caused by heat treatment or the like by acid treatment or the like. A glass substrate obtained by partially dissolving the portion is preferable.

The filter 11 preferably has heat resistance and chemical resistance. Even when heat treatment is performed in the separation operation, or when the extracellular fine particle-containing solution to be used is acidic or alkaline, such physical and chemical durability is good, so that separation and capture are stable. The operation can be performed.

(Filter manufacturing method)
The filter 11 can be manufactured by, for example, a method of manufacturing via phase separation glass or a method of forming mechanically uniform and fine through holes. Hereinafter, a method of manufacturing 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 a material used here will not be specifically limited if it is comprised from the glass which phase-separates by spinodal decomposition. Examples of such glass include SiO ₂ —B ₂ O ₃ —Na ₂ O, SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —Na ₂ O, and 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 Glass having a mother composition such as ₂ O—CaO—ZrO ₂ system, SiO ₂ —B ₂ O ₃ —Na ₂ O system, SiO ₂ —B ₂ O ₃ —CaO—MgO—Al ₂ O ₃ —TiO ₂ system, etc. Is mentioned. Among them, glass having a composition of SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —CaO—MgO—BaO—Na ₂ O system is 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. As the heating temperature is increased and the treatment time is increased, the phase separation state proceeds. As a result, a porous glass having a larger pore diameter is 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 in the range of 10 minutes to 100 hours. This condition is preferable for the borosilicate glass described above. is there.

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. 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 preferably in the range of 0.01 to 2.0 mol / L (0.01 to 2.0 N), and preferably 0.1 to 2.0 mol / L (0. It is more preferable to 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, more preferably 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, the phase separation conditions and the treatment conditions such as acid treatment may be appropriately changed so as to be used for separation of extracellular fine particles in the solution and ensure the 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).

Furthermore, to give other physical and chemical functions to the filter, for example, after making it porous, dip coating, spin coating, sputtering, ink jetting, spray coating, vapor deposition, ALD with polymer or inorganic film on the surface A predetermined functional layer may be formed on the filter surface by applying (Atomic Layer Deposition), CVD (Chemical Layer Vapor Deposition), or the like. As this functional layer, it improves the filtering performance by hydrophilic treatment to improve the hydrophilicity of the surface, gives adsorption specificity to capture only specific substances, makes it difficult to adsorb specific substances on the filter surface, A glass substrate having desired characteristics can be obtained by a functional layer to be formed or a modification treatment.

At this time, it is preferable to perform a cell non-adhesion treatment from the viewpoint of preventing impurities other than the extracellular particles to be captured from adhering to the filter. As this non-cell adhesion treatment, it is preferable to provide a protein non-adhesion layer on the filter surface using a protein adhesion inhibitor. This protein non-adhesion layer may be formed by directly applying a 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, 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 fluoropolymer having a biocompatible group described in International Publication No. 2016/002796 can be used. .

(Elastic member)
The elastic member 12 is a member provided in elastic contact with the filter 11 at the upper peripheral edge of the first main surface 11a of the filter 11 and / or the lower peripheral edge of the second main surface 11b. That is, when the elastic member 12 is provided only at the upper peripheral edge of the first main surface 11 a of the filter 11, or when the elastic member 12 is provided only at the lower peripheral edge of the second main surface 11 b of the filter 11, the first of the filter 11. In the case where it is provided on both the upper peripheral edge of the main surface 11a and the lower peripheral edge of the second main surface 11b, an embodiment is conceivable. 1A to 1C, a configuration example provided only at the upper peripheral edge of the first main surface 11a of the filter 11, and in FIG. 2, the upper peripheral edge of the first main surface 11a of the filter 11 and the peripheral edge of the second main surface 11b. Configuration examples provided in both lower portions are shown.

Here, in order to use the first main surface 11a of the filter 11 as a filtration surface, according to a normal application example, the filter 11 is disposed horizontally, the first main surface 11a is upward, and the second main surface 11b is downward. For convenience, the terms upper peripheral edge and lower peripheral edge are used, but the first main surface 11a is always facing upward and the second main surface 11b is facing downward when in use. It is not limited to cases only.

Further, “the upper peripheral edge of the first main surface 11a” means the edge of the first main surface 11a in the vicinity of the outer shape (contour shape) of the first main surface 11a when the first main surface 11a side of the filter 11 is viewed in plan view. The upper side of the position in contact with the edge is referred to as “the lower peripheral edge of the second main surface 11 b” means the outer shape (contour shape) of the second main surface 11 b in plan view of the second main surface 11 b side of the filter 11. It refers to the lower side of the position in contact with the edge (edge) of the second main surface 11b in the vicinity.

To explain further, “the elastic member 12 is provided on the upper peripheral edge of the first main surface 11a” means that the elastic member 12 is disposed so as to overlap the edge of the first main surface 11a. “The elastic member 12 is provided at the lower peripheral edge of the second main surface 11b” means that the elastic member 12 is disposed so as to overlap the edge of the second main surface 11b.

As described above, the elastic member 12 is provided at the upper peripheral edge of the first main surface 11a and / or the lower peripheral edge of the second main surface 11b. At this time, the central portions of the first main surface 11a and the second main surface 11b are provided. The shape is not covered. This is for securing the filtration surface of the filter 11. Therefore, it is preferable that the elastic member 12 has an annular shape along the outer shape of the filter 11. The outer shape is not particularly limited, such as a perfect circle, an ellipse, or a polygon, but is preferably the same as or similar to the outer shape of the filter 11.

The elastic member 12 only needs to have elasticity, and by having elasticity in this way, when an external force is applied by centrifugation or the like during filtration, the elastic member 12 and the filter 11, the elastic member 12 and the elastic member 12 will be described later. It adheres between the container used for the kit for capturing extracellular particulates to prevent the extracellular particulate-containing solution from passing between them. Thereby, the filtration efficiency can be improved.

Here, the elastic member 12, is preferably 100 ~ 50000kgf / cm ² the elastic modulus, more preferably 1000 ~ 40000kgf / cm ^2, more preferably 1000 ~ 30000kgf / cm ^2. In this specification, an elastic modulus means a bending elastic modulus and is calculated | required according to JISK7171.

Further, it is preferable that the elastic member 12 is formed of a non-permeable material having no liquid permeability. That is, the porous body and those having through-holes inside are excluded, and a non-porous body is preferable, and is typically made of a solid material. By using such an impermeable material, the extracellular member-containing solution passes from the outer periphery of the filter 11 when the elastic member 12 is combined with a container to be described later to form an extracellular particle capturing kit. It is possible to prevent effectively, and all of the extracellular particle-containing solution comes into contact with the filter 11 and is filtered. This can improve the capture efficiency of extracellular particulates.

As the material forming the elastic member 12, a known material can be used as long as it has elasticity. This material is preferably one that does not adversely affect the measurement results because it contacts the extracellular microparticles to be captured during filtration or the extracellular microparticle-containing solution containing the microparticles. More specifically, those made of a stable material that does not cause reaction or dissolution upon contact with a biological component are preferred, such as polypropylene (PP), polyethylene (PE), polyethersulfone (PES), polyethylene terephthalate (PET). Resin materials such as polycarbonate (PC), polyvinyl chloride (PVC), methacrylic resin (PMMA), and ethylenetetrafluoroethylene (ETFE), natural rubber, and synthetic rubber.

Further, the thickness of the elastic member 12 is preferably 0.1 to 3 mm, and more preferably 0.1 to 2 mm. If it is out of the above range, the elastic action is lowered or the film becomes too flexible and the degree of adhesion with the filter 11 or the container is lowered, and the filtration efficiency may be lowered.

The elastic member 12 is preferably a member having an outer shape that is slightly larger than the outer diameter shape of the filter 11 in order to improve the capture efficiency in filtration. About this modification, the schematic structure is shown in FIG. The extracellular particle capturing filter member 30 shown in FIG. 3 has a configuration in which a filter 11 and an elastic member 31 are laminated, and the outer diameter of the elastic member 31 is larger than the outer diameter of the filter 11.

At this time, the outer diameter of the elastic member 31 is preferably 1.001 to 1.1 times, more preferably 1.005 to 1.1 times the outer diameter of the filter 11. It is more preferably 0.01 to 1.08 times, and even more preferably 1.01 to 1.06 times. That is, for example, when the outer diameter of the filter 11 is 7 mm, the outer diameter of the elastic member 31 is preferably 7.007 to 7.7 mm, more preferably 7.07 to 7.56 mm, and 7.07 to 7.42 mm. Is more preferable.

1A to 3 show a configuration in which only one layer of the elastic member 12 is provided, a structure in which a plurality of layers are laminated may be used. In that case, each magnitude | size of the elastic member of a several layer may be the same, and may differ. When they are different from each other, it is preferable to laminate them so that the outer diameter becomes larger in order of lamination from the filter 11 (as the distance from the filter 11 increases).

(Adhesive layer)
As the extracellular particle capturing filter of this embodiment, an adhesive layer may be further provided between the filter 11 and the elastic member 12. By providing such an adhesive layer, the filter 11 and the elastic member 12 adhere to each other, and stable filtration can be performed even during the filtration operation, and the extracellular fine particle-containing solution can be passed from the outer periphery of the filter 11. Can also be effectively suppressed.

As an extracellular particle capturing filter provided with such an adhesive layer, FIG. 4 shows an extracellular particle capturing filter member 40 provided with an adhesive layer 41 in addition to the extracellular particle capturing filter member 10 of FIG. 1A. 5 shows an extracellular particle capturing filter member 50 provided with an adhesive layer 51 in addition to the extracellular particle capturing filter member 20 of FIG. 2, and FIG. 6 shows an extracellular particle capturing filter of FIG. The filter member 60 for capturing extracellular particulates provided with an adhesive layer 61 in addition to the member 30 is shown.

The adhesive layer used here is not particularly limited as long as it can adhere the filter 11 and the

elastic members

12 and 31, and a known adhesive layer can be used. As such an adhesive layer, for example, an adhesive layer may be applied by directly applying an adhesive on the filter 11 or the elastic member 12 and cured together with the elastic member 12 or the filter 11, or an adhesive on the support substrate. Alternatively, an adhesive layer in which the filter 11 and the elastic member 12 are bonded to each other may be used.

By adhering in this way, there is no possibility that the filter 11 and the elastic member 12 are displaced or a gap is formed between them, and a filter member for capturing extracellular particles that can be stably filtered is provided. can get.

Examples of the adhesive used here include epoxy adhesives, silicone adhesives, acrylic adhesives, vinyl acetate adhesives, urethane adhesives, and the like.

Examples of the adhesive tape used here include vinyl tape, polyester tape, polyethylene tape, polyimide tape, and the like.

[Extracellular particle capture kit]
The kit for capturing extracellular fine particles of the present embodiment is a plate having a first main surface serving as a filtration surface and a second main surface facing the first main surface, which is made of a porous body capable of capturing extracellular fine particles. A filter member for capturing extracellular particulates, and a filter member provided on the upper peripheral edge of the first main surface and / or the lower peripheral edge of the second main surface and elastically contacting the filter, and the filter member And a container for holding. That is, the filter member for capturing extracellular fine particles described above and a container for holding it are provided.

The filter member for capturing extracellular particles used here uses the filter member described above, and the description thereof is omitted.

The container used here is not particularly limited as long as it can hold the filter member for capturing extracellular particles. When this container holds the filter member for capturing extracellular particles at a predetermined position, the liquid containing the extracellular particle-containing solution to be filtered is brought into contact with the first main surface of the filter member for capturing extracellular particles. The components pass through and are discharged from the second main surface side, but the extracellular particles are captured by the extracellular particle capturing filter member and separated into solid and liquid.

Such an extracellular particle capturing kit is an extracellular particle capturing kit 70 having an extracellular particle capturing filter member 10 and a container 71 as shown in FIG. FIG. 7 shows a side sectional view of the extracellular particle capturing kit 70. Here, although the container 71 is a spin column type container, a known container that can support the filter 11 and can perform solid-liquid separation can be used. Examples of such containers include well-type containers and tube-type containers provided with a large number of filters in the plane direction.

The container 71 shown in FIG. 7 has a filter holding part 71a for holding the extracellular particle capturing filter member 10 and a containing part 71b that can contain the filtered cell fine particle-containing solution. When the container has such a configuration, the filtration of the extracellular particles captured by the extracellular particle capturing filter member 10 can be performed in more detail by removing the filtrate stored in the storage unit 71b by filtration. It can be carried out.
That is, after filtration, the accommodating part 71b is once separated from the filter holding part 71b to remove the filtrate, and the accommodating part 71b is attached to the filter holding part 71a again. Then, the contents of DNA and RNA can be discharged by dissolving the membrane of extracellular particles captured on the filter member 10 for capturing extracellular particles, and the contents can be stored in the storage portion 71b. The container 71b can be separated from the filter holder 71a again, and the contents stored in the container 71b can be easily used for measurement, analysis, and the like.

[Extracellular particle capture method]
Next, an extracellular particle capturing method to which the extracellular particle capturing kit of this embodiment is applied will be described. In the description here, the case where the extracellular particle capturing kit 70 shown in FIG. 7 is used will be described as an example.

First, in performing the extracellular particle capturing method of the present embodiment, an extracellular particle-containing solution containing extracellular particles is introduced into an extracellular particle capturing kit 70 including a filter 11 made of a porous material, Perform solid-liquid separation. By this solid-liquid separation, the liquid component of the extracellular particle-containing solution passes through the extracellular particle capturing filter member 10 and is discharged from the lower part thereof. On the other hand, extracellular particulates that are solid components cannot pass through the pores of the extracellular particulate capturing filter member 10 and are captured by at least one of the surface and the pores.

At this time, the average pore diameter of the pores of the filter 11 to be used is in the range of 30 to 500% of the average particle diameter of the extracellular fine particles, and 90% or more of the pores on a volume basis It is preferably included within the range of 60 to 140% of the peak top pore size in the pore size distribution. Further, it is more preferable that 90% or more of the pores are included in the range of 70 to 130% of the peak top pore size in the pore size distribution.

The extracellular particle-containing solution prepared here is not particularly limited as long as it contains extracellular particles to be captured and can be filtered by the above-described extracellular particle capturing filter member 10. Can do. Examples of the extracellular fine particle-containing solution include biological components such as serum, plasma, urine, and cell supernatant, and processed products thereof. At this time, it is preferable that the viscosity of the extracellular fine particle-containing solution before the separation is adjusted to 2 × 10 ⁻³ Pa · s or less so that the solid-liquid separation can be performed efficiently. Examples of the liquid component used for achieving such a viscosity include water and alcohol.

Also, this capture is usually performed by applying external force. When an external force is applied, the pressure applied to the extracellular particulate capturing filter member 10 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 filter member 10 for capturing extracellular particles can be suppressed.

In addition, it is preferable to centrifuge in order to filter by applying an external force. When centrifugation is used, it is sufficient to adopt conditions that allow efficient filtration by a known method. For example, the separation is preferably performed at a load of 100 to 15000 G for 1 to 20 minutes.

Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these descriptions. Examples 1 to 10 are examples, and example 11 is a comparative example.

[Manufacture of filters]
(Reference Example 1)
Each component of SiO ₂ , B ₂ O ₃ , Na ₂ CO ₃ as a raw material is expressed as a molar percentage on an oxide basis, and is a multi-component of SiO ₂ 65%, B ₂ O ₃ 27%, Na ₂ O 8%. The mixture was mixed so as to have a system 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 a temperature of 500 ° C. to room temperature at a rate of 30 ° C./min to obtain a glass block.

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 mm and a thickness of 1.0 mm.

Next, this plate glass was immersed in 1N HNO ₃ for 24 hours and subjected to a leaching treatment, and then washed with 1N NaOH and hot water at 60 ° C. to obtain a filter A for capturing extracellular particles. The average pore diameter of the obtained filter A was 300 nm. 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 diameter (nm)) was 0.16 cm ² / mm.

(Reference Example 2)
Glass raw materials were mixed so that a glass having a multi-component composition (SiO ₂ —Al ₂ O ₃ —B ₂ O ₃ —MgO—CaO—BaO—Na ₂ O) was obtained, and the same operation as in Reference Example 1 was performed. A glass block was obtained. The glass block was subjected to a phase separation treatment at 740 ° C. for 6 hours. Next, after processing into the same shape as in Reference Example 1, it was immersed in 1N HNO ₃ for 48 hours and leached, and then washed with 1N NaOH and 60 ° C. hot water to capture extracellular particles. Filter B was obtained. The average pore size of the obtained filter B was 600 nm. 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 diameter (nm)) was 0.14 cm ² / mm.

(Example 1)
A ring-shaped washer made of polypropylene (PP ring) having an outer diameter of 7.3 mm, an inner diameter of 4.8 mm, and a thickness of 1.5 mm is brought into contact with and arranged as an elastic member on the periphery of the filter A obtained in Reference Example 1. An outer particulate capturing filter 1 was obtained.

(Example 2)
As an elastic member, a filter 2 for capturing extracellular particles by placing and placing an O-ring washer (O-ring) made of silicon rubber having an outer diameter of 7.6 mm, an inner diameter of 3.8 mm, and a thickness of 1.9 mm on the upper and lower edges of the periphery of the filter A. Got.

(Example 3)
A silicone adhesive as an adhesive is applied to the upper peripheral edge of the filter A obtained in Reference Example 1 so that the thickness is 0.2 mm, and the PP ring is placed thereon, followed by drying and curing at 25 ° C. for 12 hours. Then, the filter A and the PP ring were joined together via an adhesive layer to obtain a filter 3 for capturing extracellular particles.

(Example 4)
On the upper peripheral edge of the filter A obtained in Reference Example 1, as an adhesive tape, a polyimide tape having an outer diameter of 7.1 mm, an inner diameter of 5.0 mm, and a thickness of 0.114 mm, and an outer diameter of 7.1 mm and an inner diameter of 4. An 8 mm, 1.5 mm thick polypropylene ring washer (PP ring) is placed, pressed between the upper and lower directions, and the filter A and PP ring are joined together via an adhesive layer. 4 was obtained.

(Examples 5 to 10)
Filter members 5 to 10 for capturing extracellular fine particles were obtained so as to have the structures described in Tables 1 and 2. Here, the adhesive layer was provided in the same manner as in Example 3, and the adhesive tape was provided in the same manner as in Example 4. Further, the adhesive layer (lower) and the elastic member (lower) provided at the lower peripheral edge were provided so that the materials and configurations used in the upper peripheral edge were the same. In particular, in Tables 1 and 2, although the diameter and thickness of the elastic member (lower) are omitted, the same elastic member (upper) provided at the upper peripheral edge of the same example was used.
In Examples 8 to 10, the filter B obtained in Reference Example 2 was used.

(Example 11)
The filter A obtained in Reference Example 1 was directly used as the filter member 11 for capturing extracellular fine particles.

(Test example)
[Measurement of exosome capture rate]
Filter members 1 to 11 for capturing extracellular fine particles obtained in Examples 1 to 11 were placed in spin columns, respectively.
As an extracellular microparticle-containing solution, a dispersion solution in which HepG2-derived exosomes labeled with a PKH26GL cell linker kit (manufactured by SIGMA-ALDRICH, trade name) is dispersed in a phosphate buffer solution (PBS) is prepared. 0.5 mL each was supplied onto the filter members 1 to 11 for capturing extracellular fine particles in the spin column. The spin column was centrifuged at 6000 G (pressure 0.8 MPa) for 10 minutes using a centrifuge, and exosomes were captured by a filter member for capturing extracellular particles. Then, the fluorescence intensity of the dispersion before and after exosome capture, that is, the dispersion before and after filtration is measured with a plate reader, and “capture rate (%) = [(fluorescence intensity before filtration−fluorescence intensity after filtration) / before filtration”. The capture rate of extracellular microparticles was calculated by the formula “fluorescence intensity] × 100”. The results are also shown in Tables 1 and 2.

From the results of the capture rates shown in Tables 1 and 2, it was found that the extracellular particle capturing filter member of this embodiment can efficiently capture extracellular particles. In addition, even if the outer diameter of the elastic member to be provided is the same as that of the filter and only the upper part of the periphery, it is possible to improve the capture rate by providing an adhesive layer (Example 4). (Examples 5 and 6).

As described above, the extracellular particle capturing filter member and extracellular particle capturing kit of the present invention can efficiently capture extracellular particles, observe and inspect the captured extracellular particles, and further, inside the extracellular particles. Analysis of contained DNA, RNA, etc. can also be performed efficiently.
It should be noted that the entire contents of the specification, claims, drawings and abstract of Japanese Patent Application No. 2018-016739 filed on February 1, 2018 are cited herein as disclosure of the specification of the present invention. Incorporated.

10, 20, 30, 40, 50, 60 ... extracellular particulate trapping filter member, 11 ... filter, 11a ... first main surface, 11b ... second main surface, 12, 31 ... elastic member, 41, 51, 61 ... adhesive layer, 70 ... extracellular particle capturing kit, 71 ... container

Claims

A plate-like filter comprising a porous body capable of capturing extracellular fine particles, and having a first main surface serving as a filtration surface and a second main surface facing the first main surface;
An elastic member provided in elastic contact with the filter at an upper peripheral edge of the first main surface and / or a lower peripheral edge of the second main surface;
A filter member for capturing extracellular particles, comprising:
The extracellular particulate capturing filter member according to claim 1, further comprising an adhesive layer that adheres the elastic member and the filter.
The extracellular particulate capturing filter member according to claim 2, wherein the adhesive layer is a ring-shaped adhesive tape provided between the elastic member and the filter.
The extracellular fine particle capturing filter member according to claim 2, wherein the adhesive layer is a resin adhesive layer provided between the elastic member and the filter.
The filter member for capturing extracellular particles according to any one of claims 1 to 4, wherein the filter has a non-protein adhesion layer on a surface thereof.
The extracellular particle capturing filter member according to any one of claims 1 to 5, wherein the elastic member is provided on each of peripheral edges of the first main surface and the second main surface.
The extracellular particle capturing filter according to any one of claims 1 to 6, wherein the elastic member has an outer peripheral diameter equal to or larger than an outer peripheral diameter of the first main surface of the filter. Element.
The filter member for capturing extracellular particulates according to any one of claims 1 to 7, wherein the elastic member has a surface in contact with the first main surface of the filter that does not contact the extracellular particulates.
The extracellular particle capturing filter member according to any one of claims 1 to 8, wherein the elastic member is impermeable to an extracellular particle-containing solution containing the extracellular particles.
The filter member for capturing extracellular fine particles according to any one of claims 1 to 9, wherein the elastic member is a non-porous body.
The filter member for capturing extracellular fine particles according to any one of claims 1 to 10, wherein the filter is an inorganic porous material.
The filter member for capturing extracellular particles according to any one of claims 1 to 11, wherein the filter has an average pore diameter of 5 to 2500 nm.
The filter member for capturing extracellular particles according to any one of claims 1 to 12, wherein the filter has a thickness of 0.1 to 3 mm.
The filter member for capturing extracellular particles according to any one of claims 1 to 13, wherein the elastic member is made of polypropylene.
The extracellular particulate capturing filter member according to any one of claims 1 to 14, wherein the elastic member has a thickness of 0.1 to 3 mm.
The extracellular particulate capturing filter member according to any one of claims 1 to 15, wherein the filter has a substantially disc shape and a diameter of 1 to 30 mm.
The extracellular member according to claim 16, wherein the elastic member is substantially ring-shaped, and an outer peripheral diameter of the elastic member is equal to or larger by 0.1% to 10% than an outer peripheral diameter of the filter. Filter member for capturing fine particles.
A plate-like filter comprising a porous body capable of capturing extracellular particulates, having a first main surface serving as a filtration surface and a second main surface facing the first main surface, and an upper peripheral edge of the first main surface And / or an elastic member provided in elastic contact with the filter at the lower peripheral edge of the second main surface, and a filter member for capturing extracellular particulates;
And a container for holding the filter member.
The extracellular particle capturing kit according to claim 18, wherein the container is a spin column type or a well type.
An extracellular particle-containing solution containing extracellular particles is brought into contact with the extracellular particle capturing kit according to claim 18 or 19,
Filtering the extracellular particle-containing solution using external force, and capturing the extracellular particles on the filter member of the extracellular particle capturing kit;
A method for capturing extracellular microparticles.