JP7660870B2 - Method and apparatus for separating and recovering particles - Google Patents

Description

The present invention relates to a novel method for separating and recovering particles such as extracellular vesicles and viruses, and a particle separation and recovery device that can be suitably used to carry out the method.

In 1983, it was discovered that vesicles with a diameter of about 30 nm to 200 nm, which are composed of lipid bilayer membranes, were secreted from reticulocytes, and were named exosomes (see Non-Patent Document 1). Around the time of the discovery of exosomes, it was discovered that various cells secrete membrane vesicles of different sizes, etc., and they are called by various names, but the International Society for Extracellular Vesicles (ISEV), an international research society for vesicles, recommends the use of extracellular vesicles as a general term for vesicles secreted from these cells. It is becoming clear that extracellular vesicles, including exosomes, transport various physiologically active substances while moving between cells. In multicellular organisms, interactions between cells are involved in various vital activities, and their breakdown leads to various diseases. Therefore, it is expected that elucidation of intercellular interactions involving extracellular vesicles will lead to an understanding of the molecular mechanisms behind various vital activities, an understanding of the pathology of various diseases, and the development of new diagnostic and therapeutic methods.

Research on exosomes generally proceeds through a series of steps: (i) preparation of samples containing exosomes, such as serum, plasma, cell culture supernatant, milk, urine, semen, cerebrospinal fluid, saliva, and tears; (ii) recovery and purification of exosomes from the samples; and (iii) confirmation of the recovered exosomes (confirmation of size, shape, etc., confirmation of exosome markers, etc.) or experiments using the recovered exosomes (searching for and analysis of biomarkers contained in exosomes, analysis of physiological functions and mechanisms of action, etc.).

The most important of the above steps is the recovery and purification of exosomes. Ultracentrifugation can recover exosomes with a size of about 100 nm, and has been widely used as a standard method. However, ultracentrifugation can only process a small amount of biological sample per run. Therefore, in order to recover exosomes from a large amount of biological sample, the biological sample must be divided into small portions and the operation must be performed multiple times, which is time-consuming and costly. In addition, expensive equipment is required, and skilled techniques are required to achieve satisfactory reproducibility of concentration and recovery efficiency. To compensate for the shortcomings of ultracentrifugation, methods such as precipitation by adding volume-exclusion polymers such as PEG and PVA, size exclusion chromatography, ultrafiltration, and separation by adding magnetic particles have been proposed (see Patent Documents 1 to 3). However, size exclusion chromatography, ultrafiltration, and precipitation methods using PEG, etc. inevitably result in contamination with contaminants other than the target exosomes (e.g., relatively large vesicles such as apoptotic bodies and high molecular weight proteins, etc.), and methods using magnetic particles have problems such as recovering exosomes with different properties than those recovered by ultracentrifugation.

International Publication No. 2013/158203 JP 2013-188212 A Special Publication No. 2012-533308

Johnstone, R. M., Adam, M., Hammond, J. R. et al.: Vesicle formation during reticulocyte maturation. Association of membrane plasma activities with released vesicles (exosomes). J. Biol. Chem., 262, 9412-9420 (1987)

However, ultracentrifugation and methods using magnetic particles can only process a small amount of biological sample per run. Therefore, in order to recover exosomes from a large amount of biological sample, the biological sample must be divided into small portions and the procedure must be repeated multiple times, which is problematic in terms of time and cost. In particular, ultracentrifugation requires expensive equipment and skilled techniques to achieve satisfactory reproducibility in concentration and recovery efficiency. In addition, ultrafiltration and precipitation methods using PEG, etc., have problems such as the inevitable contamination with contaminants other than the target exosomes (e.g., high molecular weight proteins, etc.).

The present invention has been made in consideration of these circumstances, and aims to provide a highly efficient and high-throughput particle separation and recovery method that can inexpensively and easily separate and recover particles such as exosomes with sizes between 30 nm and 10 μm from a sample solution, and a particle separation and recovery device that can be suitably used for this method.

In accordance with the above objective, the first aspect of the present invention solves the above problem by providing a method for separating and recovering particles, the method including the steps of preparing a sample solution containing particles with sizes between 30 nm and 10 μm, passing the sample solution through a track-etched membrane, and collecting the particles in the sample solution on the track-etched membrane.

A second aspect of the invention is a method for detecting particles in a sample comprising:
a downstream chamber in communication with the upstream chamber;
The above problem is solved by providing a particle separation and recovery device having a track-etched membrane for collecting the particles in the sample solution, which is positioned between the upstream chamber and the downstream chamber so as to enable the sample solution to pass from the upstream chamber to the downstream chamber.

In the particle separation and recovery method according to the first aspect of the present invention and the particle separation and recovery device according to the second aspect of the present invention, it is preferable that the pore size of the track-etched membrane is 10 nm or more and 10 μm or less.

In the particle separation and recovery method according to the first aspect of the present invention and the particle separation and recovery device according to the second aspect of the present invention, the material of the track-etched membrane is preferably any one of polytetrafluoroethylene, polyvinylidene fluoride, polyethersulfone, mixed cellulose ester, polyamide, polyester, polypropylene, polyethylene, polycarbonate, polyester, and polyimide.

In the particle separation and recovery method according to the first aspect of the present invention and the particle separation and recovery device according to the second aspect of the present invention, it is preferable that the difference between the maximum and minimum pore diameters of the track-etched membrane is within 20% of the maximum value.

In the particle separation and recovery method according to the first aspect of the present invention, the particles may be extracellular vesicles.

In the particle separation and recovery method according to the first aspect of the present invention, the particles may be exosomes.

The present invention provides a highly efficient and high-throughput particle separation and recovery method that can inexpensively and easily separate and recover particles such as exosomes with sizes between 30 nm and 10 μm from a sample solution, and a particle separation and recovery device that can be suitably used for the method.

1 is a schematic diagram of a particle separation and recovery device according to an embodiment of the present invention. 1A and 1B are schematic structural diagrams of a particle separation and recovery device according to one embodiment of the present invention, in which (a) shows the upstream member and the downstream member separated, (b) shows the two members assembled, and (c) shows the top surface of the downstream member. 1A is a schematic structural diagram of a particle separation and recovery device according to one embodiment of the present invention, in which (a) shows the upstream member, the centrifuge tube, and the lid separated, (b) shows the three components assembled, and (c) shows a modified example of the upstream member. 1 is a graph showing the measurement results of the zeta potential of exosomes separated and recovered by Example 1 and Comparative Example. 1 is a graph showing the quantification results of CD63 in exosomes separated and recovered by Example 1 and Comparative Example.

The particle separation and recovery method according to the first embodiment of the present invention (hereinafter, sometimes abbreviated as "particle separation and recovery method" or "separation and recovery method") includes the steps of preparing a sample solution containing particles with sizes of 30 nm to 10 μm, and passing the sample solution through a track-etched membrane and collecting particles in the sample solution on the track-etched membrane.

The particles to be separated and collected are between 30 nm and 10 μm in size, and specific examples include exosomes, apoptotic bodies, extracellular vesicles such as microvesicles, viruses, and nanoparticles.

Examples of sample solutions containing particles such as those described above include cell culture supernatants containing extracellular vesicles and viruses, biological samples such as body fluids (serum, plasma, milk, urine, semen, cerebrospinal fluid, saliva, tears, etc.), and reaction mixtures for nanoparticle synthesis reactions. These solutions may be used as sample solutions as they are, but if necessary, they may be pretreated using a membrane filter or the like to remove impurities (cells, etc.) other than the particles to be separated and recovered.

Track-etched membranes, which are used to separate and recover particles from a sample solution, are formed by irradiating a polymer membrane with a heavy ion beam using a cyclotron or the like to form tracks of heavy ions randomly distributed so as to penetrate the membrane from one side to the other side of the membrane, and then selectively dissolving the tracks using a technique called wet chemical etching, resulting in a porous polymer membrane in which a large number of pores with a narrow pore size distribution are arranged almost parallel to each other. In the case of porous membranes with a mesh-like structure that have been used conventionally as precision filtration membranes, there is a risk that particles are captured within the matrix that forms the mesh structure, resulting in a decrease in recovery rate or separation efficiency, whereas in the case of track-etched membranes, there is no risk of particles being captured inside the matrix. In addition, by controlling the conditions for forming the tracks by irradiation with a heavy ion beam, the pore size, pore distribution, spacing between adjacent pores, aperture ratio, etc. of the pores formed on the membrane can be controlled, making it easy to optimize the membrane according to the particles to be separated and recovered.

The pore size of the track-etched membrane used for particle separation and recovery is appropriately selected according to the size of the particles to be separated and recovered, but a pore size in the range of 10 nm to 10 μm that does not allow particles to pass through the pores is appropriately selected. It is preferable that the difference between the maximum and minimum pore sizes is within 20% of the maximum value. If the pore size distribution (difference between the maximum and minimum pore sizes) exceeds the aforementioned range, the particle recovery efficiency decreases, which is not preferable.

Specific examples of materials for track-etched membranes include polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), mixed cellulose ester (MCE), polyamide (nylon, etc.), polyester (PET, PBT, etc.), polypropylene (PP), polyethylene (PE: LDPE, HDPE, etc.), polycarbonate, polyimide, etc.

The distribution of pores on the track-etched membrane, the density of pores, the number of pores (opening ratio), the material, area and thickness of the track-etched membrane are appropriately selected according to the conditions of use, such as the type and size of particles, the volume of the sample solution and the type of solvent.

Separation and recovery of particles in a sample solution using a track-etched membrane is performed by passing the sample solution through the track-etched membrane and collecting the particles on the track-etched membrane. Any known device can be used for separating and collecting particles, but for example, a particle separation and recovery device (hereinafter sometimes abbreviated as "separation and recovery device") 10 according to a second embodiment of the present invention having a schematic structure as shown in Figure 1 is preferably used. The particle separation and recovery device 10 has an upstream chamber 11 for receiving a sample solution containing particles, a downstream chamber 12 communicating with the upstream chamber, and a track-etched membrane 13 for collecting particles in the sample solution, which is arranged between the upstream chamber and the downstream chamber so that the sample solution can be passed from the upstream chamber to the downstream chamber.

As for the structure of the separation and recovery device, any known structure such as a separation and recovery device including a membrane filter, a filtration device, etc. can be applied without any particular restrictions, as long as the track-etched membrane can be used as a separation and recovery material. For example, as shown in FIG. 2, the separation and recovery device 20 may be composed of a cylindrical upstream member 21 having openings at both ends and a hollow cylindrical downstream member 22 (see FIG. 2(b)). The diameter of the upstream member 21 is smaller than the diameter of the downstream member 22. A gasket 24 made of an elastic material such as rubber is provided at the malleable side opening of the upstream member 21. An engagement protrusion 26 is formed on the upper surface side of the downstream member 22, which engages with the upstream member 21 (see FIG. 2(a)), holds both liquid-tight via the gasket 24, and holds the track-etched membrane 23. A membrane holder cover 25 having a window and detachable is attached to the upper surface of the engagement protrusion 26 of the downstream member 22. When the membrane holder lid 25 is removed, a disk-shaped recess (not shown) with a window is formed so that part of the track-etched membrane 23 is exposed. By placing the circular track-etched membrane 23 and attaching the membrane holder lid 25 again, the track-etched membrane 23 can be held on the upper surface of the downstream member 22 (see FIG. 2(c)). A pressure reduction port 27 is provided on the side of the downstream member 22, and by attaching a pressure reduction means such as a pressure reduction pump to this port, the inside of the downstream member 22 can be reduced in pressure, and the flow rate of the sample solution can be increased due to the pressure difference with the upstream member 21 side.

As shown in FIG. 2(b), by attaching an upstream member 21 to a downstream member 22 holding a track-etched membrane 23, a separation and recovery device 20 is formed, which has an upstream chamber (a space defined by the upstream member 21 and the upper surface of the engagement protrusion 26) for receiving a sample solution containing particles such as exosomes, a downstream member (downstream chamber 22) communicating with the upstream chamber, and a track-etched membrane 23 for collecting particles in the sample solution, which is disposed between the upstream chamber and the downstream chamber so that the sample solution can be passed from the upstream chamber to the downstream chamber.

In the downstream member 22 shown in FIG. 2, the engagement protrusion 26 is integrally molded, but it may be configured to be separable as a main body and a lid. Also, instead of providing a pressure reduction port 27, a pressurizing means may be provided on the upstream member 21 side to increase the flow rate of the sample solution by pressurizing the upstream chamber. Pressurization of the upstream chamber and depressurization of the downstream chamber may be applied in combination.

As shown in FIG. 3, the separation and recovery device 30 may be configured as an upstream member 31, which is a cylindrical member having a smaller diameter than the centrifuge tube and a track-etched membrane 33 integrally molded on the lower side, and which has a flange 34 for holding the upper end of the centrifuge tube 32, as shown in FIG. 3(a). The upstream member 31 is inserted from the opening of the centrifuge tube 32, and is held at the upper end of the opening of the centrifuge tube 32 by the flange 34, and a lid 35 is attached, thereby forming the separation and recovery device 30 (see FIG. 3(b)). Before attaching the lid 35, the sample solution is poured into the upstream member 31, and the lid 35 is closed and centrifuged, so that particles in the sample solution are collected on the track-etched membrane 33.

As shown in Figures 3(a) and 3(b), the track-etched membrane 33 may be formed in a cone-like cross-sectional shape that is convex downward, or as shown in Figure 3(c), it may be formed in an inclined ellipse or a circle that is approximately perpendicular to the wall surface.

Next, examples carried out to confirm the effects of the present invention will be described.
Example 1: Separation and recovery of exosomes from cell culture supernatant using a track-etched membrane Using an apparatus as shown in FIG. 2, a track-etched membrane with a diameter of 90 mm (it4ip, made of polycarbonate, 1000M25/911N101/A4-5, maximum pore size 0.1 μm) was set in the membrane holder, and 15 mL of PBS (phosphate buffered saline) was passed through under reduced pressure. Next, 50 mL of cell culture supernatant (serum-free medium, HEK293s) that had been passed through a 0.22 μm syringe filter was passed through under reduced pressure. 15 mL of PBS was passed through under reduced pressure, and the particles remaining on the membrane were collected with a small amount of PBS.

Comparative Example: Separation and Recovery of Exosomes from Cell Culture Supernatant Using Ultracentrifugation For comparison, the cell culture supernatant (same as above) passed through a 0.22 μm syringe filter was subjected to ultracentrifugation at 100,000 × g for 2 hours. The precipitate was washed with PBS, and then ultracentrifuged again at 100,000 × g for 2 hours. The precipitate that had accumulated at the bottom of the centrifuge tube was collected with a small amount of PBS.

Evaluation of Separated and Recovered Exosomes For the exosomes separated and recovered from the cell culture supernatant in Example 1 and Comparative Example, the zeta potential was measured and the amount of CD63, a marker protein in exosomes, was quantified.

Zeta potential was measured by suspending 0.3 μg of exosomes in 50 mL HEPES buffer 1 mL and using a zeta potential measuring device (Zetasizer). Quantitation of CD63 was performed by immunostaining using PS Capture Exosome ELISA Kit manufactured by Fujifilm Wako Pure Chemical Industries. The measurement results of zeta potential and the quantification results of CD63 of exosomes separated and recovered in Example 1 and Comparative Example are shown in Figures 4 and 5, respectively. In Figures 4 and 5, "UC" shows the measurement results of exosomes separated and recovered in Comparative Example (using ultracentrifugation method) and "TEM" shows the measurement results of exosomes separated and recovered in Example 1 (using track-etched membrane). As is clear from Figures 4 and 5, the results of both were almost the same. The fluctuation of zeta potential due to adhesion of polymer to the surface observed for exosomes separated and recovered by precipitation method using volume exclusion polymer was not observed in exosomes separated and recovered in Example 1. Furthermore, the quantitative results for CD63 were comparable for both methods, and no adsorption of exosomes onto the track-etched membrane was observed. These results confirmed that the exosome separation and recovery method in Example 1 allows the easy production of high-purity exosomes comparable to those obtained by ultracentrifugation.

10, 20, 30: Particle separation and recovery device 11: Upstream chamber 12: Downstream chamber 13, 23, 33, 33a, 33b: Track-etched membrane 21, 31, 31a, 31b: Upstream member 22: Downstream member 24: Gasket 25: Membrane holder lid 26: Engagement protrusion 27: Pressure reduction port 32: Centrifuge tube 34: Flange 35: Lid

Claims (8)

Preparing a sample solution containing exosomes ;
and passing the sample solution through a track-etched membrane and collecting the exosomes in the sample solution on the track-etched membrane.
The method for separating and recovering exosomes , wherein the track-etched membrane is made of polycarbonate . The method for separating and recovering exosomes according to claim 1, characterized in that the pore size of the track-etched membrane is 10 nm or more and 10 μm or less. The method for separating and recovering exosomes according to claim 1 or 2, characterized in that the difference between the maximum and minimum pore diameters of the track-etched membrane is within 20% of the maximum value. An upstream chamber for receiving a sample solution containing exosomes ;
a downstream chamber in communication with the upstream chamber;
a track-etched membrane for collecting the exosomes in the sample solution, the track-etched membrane being disposed between the upstream chamber and the downstream chamber so as to allow the sample solution to pass from the upstream chamber to the downstream chamber;
The exosome separation and recovery device , wherein the material of the track-etched membrane is polycarbonate . The exosome separation and recovery device according to claim 4 , characterized in that the pore diameter of the track-etched membrane is 10 nm or more and 10 μm or less. The exosome separation and recovery device according to claim 4 or 5 , characterized in that the difference between the maximum and minimum pore diameters of the track-etched membrane is within 20% of the maximum value. an upstream member that is a component of the upstream chamber and a downstream member that is a component of the downstream chamber,
the upstream member has openings at both ends and a packing made of an elastic member is provided at the opening at the lower end,
The exosome separation and recovery device according to any one of claims 4 to 6, wherein the downstream member has an engagement protrusion on its upper surface side that engages with the upstream member and holds both of them liquid-tight via the packing , and that holds the track- etched membrane. The upstream chamber includes an upstream member, the downstream chamber includes a centrifuge tube, and a lid attached to the centrifuge tube;
the upstream member is a cylindrical member having a flange for holding the upper end of the centrifuge tube, the cylindrical member having a diameter smaller than that of the centrifuge tube, and the track-etched membrane is integrally formed on the lower side of the cylindrical member;
The exosome separation and recovery device according to any one of claims 4 to 6 , wherein the lid is attached to the centrifuge tube after the upstream member is inserted from the opening of the centrifuge tube and the flange holds the lid at the upper end side of the opening of the centrifuge tube .