WO2022004759A1 - Particle separation and recovery method and particle separation and recovery apparatus - Google Patents

Abstract

Disclosed is a particle separation and recovery method which includes a step for preparing an exosome-containing sample solution and a step for passing the sample solution through a track-etched membrane and collecting particles contained in the sample solution on the track-etched membrane. Further disclosed is a particle separation and recovery apparatus 10 which has an upstream chamber 11 for receiving an exosome-containing sample solution, a downstream chamber 12 which is connected to the upstream chamber, and a track-etched membrane 13 which collects particles contained in the sample solution and is positioned between the upstream chamber 11 and the downstream chamber 12 so as to allow passage of the sample solution therethrough from the upstream chamber 11 to the downstream chamber 12.

Description

Particle separation and recovery method and particle separation and recovery device

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

In 1983, it was discovered that vesicles with a diameter of about 30 nm to 200 nm consisting of a lipid bilayer membrane 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, and they are called by various names, but it is an international research society for extracellular vesicles. The International Society for Extracellular Vesicles (ISEV) recommends the use of extracellular vesicles as a general term for vesicles secreted from these cells. It is becoming clear that extracellular vesicles such as exosomes transport various bioactive substances while moving between cells. In multicellular organisms, cell-cell interactions are involved in various life activities, and their breakdown leads to various diseases. Therefore, elucidation of cell-cell interactions involving extracellular vesicles is a variety of life activities. It is expected to lead to an understanding of the molecular mechanism behind the disease, an understanding of the pathophysiology of various diseases, and the development of new diagnostic and therapeutic methods.

Studies on exosomes generally include (i) preparation of samples containing exosomes such as serum, plasma, cell culture supernatant, milk, urine, semen, cerebrospinal fluid, saliva, and tears, and (ii) recovery of exosomes from samples. And purification, (iii) confirmation of recovered exosomes (confirmation of size, shape, etc., confirmation of exosome markers, etc.) or experiments using recovered exosomes (search and analysis of biomarkers contained in exosomes, physiological functions and actions). It proceeds through a series of steps (analysis of mechanism, etc.).

The most important of the above steps is the recovery and purification of exosomes. The ultracentrifugal method has been widely used as a standard method because it can recover exosomes having a size of about 100 nm. However, the ultracentrifugal method requires a small amount of biological sample to be processed at one time. Therefore, in order to recover exosomes from a large amount of biological samples, it is necessary to divide the biological samples into small pieces and perform operations a plurality of times, which causes a problem that a lot of time and cost are required. Further, there is a problem that an expensive device is required and skill is required to satisfy the reproducibility of concentration and recovery efficiency. As a method for compensating for the shortcomings of the ultracentrifugation method, a method of precipitating by adding a volume exclusion polymer such as PEG or PVA, a size exclusion chromatography method, a limit filtration method, a method of separating by adding magnetic particles, etc. have been proposed so far. (See Patent Documents 1 to 3). However, in the size exclusion chromatography method, the ultracentrifuge filtration method, the precipitation method by PEG, etc., impurities other than the target exosome (for example, relatively large vesicles such as extracellular vesicles and high molecular weight proteins, etc.) are used. ) Is inevitable, and the method using magnetic particles has problems such as recovery of exosomes having different properties from the ultracentrifugation method.

International Publication No. 2013/158203 Pamphlet Japanese Unexamined Patent Publication No. 2013-188212 Special Table 2012-533308 Gazette

However, with the ultracentrifugation method and the method using magnetic particles, the amount of biological sample processed at one time is small. Therefore, in order to recover exosomes from a large amount of biological samples, it is necessary to divide the biological samples into small pieces and perform operations a plurality of times, which causes a problem that a lot of time and cost are required. In particular, the ultracentrifugation method requires expensive equipment and skill is required to satisfy the reproducibility of concentration and recovery efficiency. In addition, the ultrafiltration method and the precipitation method using PEG or the like have problems that contamination with impurities other than the target exosome (for example, high molecular weight protein) cannot be avoided.

The present invention has been made in view of such circumstances, and it is possible to separate and recover particles having a size of 30 nm to 10 μm such as exosomes from a sample solution inexpensively and easily, and to separate and recover highly efficient and high-throughput particles. It is an object of the present invention to provide a method and a particle separation / recovery device that can be suitably used for the method.

The first aspect of the present invention according to the above object is a step of preparing a sample solution containing particles having a size of 30 nm to 10 μm, and a track-etched membrane in which the sample solution is passed and placed on the track-etched membrane. The above problem is solved by providing a method for separating and recovering particles including a step of collecting the particles in the sample solution.

A second aspect of the present invention comprises an upstream chamber for receiving a sample solution containing particles having a size of 30 nm to 10 μm.
A downstream chamber communicating with the upstream chamber,
Collect the particles in the sample solution 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. The above problem is solved by providing a particle separation / recovery device having a track-etched chamber for the purpose of the above-mentioned.

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

In the method for separating and recovering particles according to the first aspect of the present invention and the device for separating and recovering particles according to the second aspect of the present invention, the material of the track-etched membrane is polytetrafluoroethylene, polyvinylidene fluoride, or poly. It is preferably any one of ether sulfone, cellulose mixed ester, polyamide, polyester, polypropylene, polyethylene, polycarbonate, polyester and polyimide.

In the particle separation / recovery method according to the first aspect of the present invention and the particle separation / recovery device according to the second aspect of the present invention, the difference between the maximum value and the minimum value of the pore diameter of the track-etched membrane is the above. It is preferably within 20% of the maximum value.

In the method for separating and recovering particles according to the first aspect of the present invention, the particles may be extracellular vesicles.

In the method for separating and recovering particles according to the first aspect of the present invention, the particles may be exosomes.

According to the present invention, it is suitably used for a highly efficient and high-throughput particle separation / recovery method capable of separating and recovering particles having a size of 30 nm to 10 μm such as exosomes from a sample solution inexpensively and easily. A particle separation and recovery device capable of producing particles is provided.

It is a schematic diagram of the particle separation recovery apparatus which concerns on one Embodiment of this invention. It is a schematic structural diagram of the particle separation and recovery apparatus which concerns on one Embodiment of this invention, (a) shows the state which the upstream side member and the downstream side member were separated, and (b) is the state which both were assembled. (C) shows the upper surface of the downstream member. It is a schematic structural diagram of the particle separation and recovery apparatus which concerns on one Embodiment of this invention, (a) shows the state which the upstream member, the centrifugal tube and the lid are separated, and (b) is the state which assembled the three. (C) shows a modified example of the upstream member. It is a graph which shows the measurement result of the zeta potential of the exosome separated and recovered by Example 1 and the comparative example. It is a graph which shows the quantitative result of the exosome CD63 separated and recovered by Example 1 and the comparative example.

The method for separating and recovering particles according to the first embodiment of the present invention (hereinafter, may be abbreviated as "separation and recovery method for particles", "separation and recovery method", etc.) is a particle having a size of 30 nm to 10 μm. It includes a step of preparing a sample solution containing the above, and a step of 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 recovered are particles having a size of 30 nm to 10 μm, and specific examples thereof include exosomes, extracellular vesicles, extracellular vesicles such as microvesicles, viruses, nanoparticles, and the like.

Sample solutions containing the above particles include extracellular vesicles, cell culture supernatants containing viruses, and living organisms such as body fluids (serum, plasma, milk, urine, semen, cerebrospinal fluid, saliva, tears, etc.). Examples thereof include a reaction mixture of a sample and a synthetic reaction of nanoparticles. These solutions may be used as sample solutions as they are, but if necessary, pretreatment is performed using a membrane filter or the like in order to remove impurities (cells, etc.) other than the particles to be separated and recovered. You may.

The track-etched membrane used for separating and recovering particles from a sample solution irradiates a polymer membrane with an ion beam of heavy ions using a cyclotron or the like, and penetrates the membrane from one side to the other side of the membrane. A large number of fine particles with a narrow pore size distribution are formed by forming trajectories (tracks) of randomly distributed heavy ions, and then selectively dissolving the track portions using a technique called wet chemical etching. A porous polymer membrane in which pores are arranged so as to be arranged substantially in parallel. In the case of a porous membrane having a network structure, which has been conventionally used as a microfiltration membrane, particles may be collected in the matrix forming the network structure, and the recovery rate may decrease or the separation efficiency may decrease. On the other hand, in the case of the track-etched membrane, there is no possibility that particles are collected inside the matrix. In addition, by controlling the conditions for forming the track by irradiating the heavy ion beam, it is possible to control the pore diameter of the pores formed on the membrane, the distribution of the pores, the spacing between adjacent pores, the aperture ratio, etc. It is easy to perform optimization according to the particles to be recovered.

The pore diameter of the track-etched membrane used for separation and recovery of particles is appropriately selected according to the size of the particles to be separated and recovered, but the particles do not pass through the pores in the range of 10 nm or more and 10 μm or less. The pore diameter is appropriately selected. Regarding the distribution of the pore diameter, it is preferable that the difference between the maximum value and the minimum value of the pore diameter is within 20% of the maximum value. If the distribution of the pore diameter (difference between the maximum value and the minimum value of the pore diameter) exceeds the above-mentioned range, the particle recovery efficiency is lowered, which is not preferable.

Specific examples of the material of the truck-etched membrane include polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyether sulfone (PES), cellulose mixed ester (MCE), polyamide (nylon, etc.), polyester (PET). , PBT, etc.), polypropylene (PP), polyethylene (PE: LDPE, HDPE, etc.), polycarbonate, polyimide, etc.

Regarding the distribution of pores on the track-etched membrane, the density of pores, the number of pores (aperture ratio), the material, area and thickness of the track-etched membrane, etc., the type, size and volume of the sample solution , It is appropriately selected according to the usage conditions such as the type of solvent.

Separation and recovery of particles in the sample solution using the 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 the separation and recovery of particles. For example, a particle separation and recovery device according to a second embodiment of the present invention having a schematic structure as shown in FIG. 1 (hereinafter referred to as a particle separation and recovery device). , "Separation and recovery device" may be abbreviated.) 10 is preferably used. The particle separation / recovery device 10 is provided from the upstream chamber 11 between the upstream chamber 11 for receiving the sample solution containing the particles, the downstream chamber 12 communicating with the upstream chamber, and the upstream chamber and the downstream chamber. It has a track-etched membrane 13 for collecting particles in the sample solution, which is arranged so that the sample solution can be passed through the downstream chamber.

As the structure of the separation / recovery device, any known structure such as a separation / recovery device including a membrane-like filter, a filtration device, etc. may be applied without particular limitation as long as the truck-etched membrane can be used as the separation / recovery material. can. For example, as shown in FIG. 2, the separation / recovery device 20 may be composed of a cylindrical upstream member 21 having openings at both ends and a hollow cylindrical downstream member 22 (FIG. 2 (b). )reference). The diameter of the upstream member 21 is smaller than the diameter of the downstream member 22. A packing 24 made of an elastic member such as rubber is provided in the malleable side opening of the upstream member 21. The upper surface side of the downstream member 22 is engaged with the upstream member 21 (see FIG. 2A) to hold both liquid-tightly via the packing 24 and to hold the track-etched membrane 23. The engaging convex portion 26 of the above is formed. A removable membrane holder lid 25 has a window on the upper surface of the engaging convex portion 26 of the downstream member 22. When the membrane holder lid portion 25 is removed, a disk-shaped recess (not shown) provided with a window portion is formed so that a part of the track-etched membrane 23 is exposed. Therefore, the circular track-etched membrane 23 is formed. The track-etched membrane 23 can be held on the upper surface of the downstream member 22 by mounting the membrane holder lid portion 25 again (see FIG. 2C). A decompression port 27 is provided on the side surface of the downstream member 22, and by attaching a decompression means such as a decompression pump, the inside of the downstream member 22 is decompressed and the pressure difference from the upstream member 21 side. Therefore, the flow rate of the sample solution can be increased.

As shown in FIG. 2B, by attaching the upstream member 21 to the downstream member 22 holding the track-etched membrane 23, an upstream chamber (upstream side) for receiving a sample solution containing particles such as exosomes is received. From the upstream chamber, between the member 21 and the downstream member (downstream chamber 22) communicating with the upstream chamber, and between the upstream chamber and the downstream chamber (the space defined by the upper surface of the engaging protrusion 26). A separation / recovery device 20 having a track-etched membrane 23 for collecting particles in the sample solution, which is arranged so that the sample solution can be passed through the downstream chamber, is configured.

In the downstream member 22 shown in FIG. 2, the engaging convex portion 26 is integrally molded, but it may be configured to be separable as a main body portion and a lid portion. Further, instead of providing the decompression port 27, a pressurizing means may be provided on the upstream side member 21 side to pressurize the upstream chamber to increase the flow rate of the sample solution. Both pressurization of the upstream chamber and depressurization of the downstream chamber may be applied in combination.

Further, as shown in FIG. 3, the separation / recovery device 30 uses the centrifuge tube 32 as a component of the downstream chamber, and the upstream chamber has a flange 34 for holding the centrifuge tube 32 at the upper end. , A cylindrical member having a diameter smaller than that of the centrifugal tube, and may be configured as an upstream member 31 in which a track-etched membrane 33 is integrally molded on the lower side (see FIG. 3A). The separation / recovery device 30 is configured by inserting the upstream member 31 through the opening of the centrifuge tube 32, holding it on the upper end side of the opening of the centrifuge tube 32 with the flange 34, and attaching the lid 35 (FIG. 3 (b). )reference). Before attaching the lid 35, the sample solution is poured into the upstream member 31, the lid 35 is closed and centrifugation is performed, so that the particles in the sample solution are collected on the track-etched membrane 33.

As shown in FIGS. 3 (a) and 3 (b), the track-etched membrane 33 may be formed in a conical shape whose cross-sectional shape is convex downward, but FIG. 3 ( As shown in c), it may be formed in an inclined elliptical shape or a circular shape substantially perpendicular to the wall surface.

Next, an example carried out for confirming the action and effect of the present invention will be described.
Example 1: Separation and recovery of exosomes from cell culture supernatant using a track-etched membrane Using a device as shown in FIG. 2, a track-etched membrane having a diameter of 90 mm (it4ip, made of polycarbonate, 1000M25 / 911N101 / A4). -5, maximum pore diameter 0.1 μm) was set in the membrane holding portion, and 15 mL of PBS (phosphate buffered saline) was passed under reduced pressure conditions. Then, 50 mL of the cell culture supernatant (serum-free medium, HEK293s) passed through a 0.22 μm syringe filter was passed under reduced pressure conditions. 15 mL of PBS was passed under reduced pressure conditions, and the particles remaining on the membrane were recovered with a small amount of PBS.

Comparative example: Separation and recovery of exosomes from cell culture supernatant using ultracentrifugation For comparison, 100,000 × g of cell culture supernatant (same as above) passed through a 0.22 μm syringe filter. The cells were subjected to ultracentrifugation treatment for 2 hours. The precipitate was washed with PBS and then ultracentrifuged again at 100,000 xg for 2 hours. The precipitate collected at the bottom of the centrifuge tube was collected with a small amount of PBS.

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

The zeta potential was measured by suspending 0.3 μg of exosomes in terms of protein amount in 50 mL HEPES buffer 1 mL and using a zeta potential measuring device (Zetasizer). The quantification of CD63 was performed by an immunostaining method using PS Capture Exosome ELISA Kit manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. The measurement results of the zeta potential of the exosomes separated and recovered in Example 1 and Comparative Example and the quantitative results of CD63 are shown in FIGS. 4 and 5, respectively. In FIGS. 4 and 5, "UC" shows the measurement results for comparative examples (using the ultracentrifugation method), and "TEM" shows the measurement results for the exosomes separated and recovered by Example 1 (using the track-etched membrane). As is clear from FIGS. 4 and 5, the results of both were almost the same. No change in the zeta potential due to the adhesion of the polymer to the surface observed for the exosomes separated and recovered by the sedimentation method using the volume exclusion polymer or the like was not observed in the exosomes separated and recovered by Example 1. In addition, the quantitative results of CD63 were similar to each other, and no adsorption of exosomes on the track-etched membrane was observed. From these results, it was confirmed that the separation and recovery of exosomes in Example 1 can obtain high-purity exosomes comparable to the ultracentrifugation method by a simple operation.

It should be noted that the present invention enables various embodiments and modifications without departing from the broad spirit and scope of the present invention. Further, the above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the invention is indicated by the claims, not by embodiments. And various modifications made within the scope of the claims and within the equivalent meaning of the invention are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2020-112203 filed on June 30, 2020, and includes the specification, claims, drawings and abstract. The disclosure in the above Japanese patent application is incorporated herein by reference in its entirety.

10, 20, 30: Particle separation / recovery device 11: Upstream chamber 12: Downstream chamber 13, 23, 33, 33a, 33b: Track-etched membrane 21, 31, 31a, 31b: Upstream member 22: Downstream side Member 24: Packing 25: Chamber holder lid 26: Engagement convex 27: Decompression port 32: Centrifugal tube 34: Flange 35: Lid

Claims (10)

1. A step of preparing a sample solution containing particles having a size of 30 nm to 10 μm, and
   A method for separating and recovering particles, which comprises a step of passing the sample solution through a track-etched membrane and collecting the particles in the sample solution on the track-etched membrane.
2. The method for separating and recovering particles according to claim 1, wherein the pore diameter of the track-etched membrane is 10 nm or more and 10 μm or less.
3. Claim 1 is characterized in that the material of the track-etched membrane is any one of polytetrafluoroethylene, polyvinylidene fluoride, polyether sulfone, cellulose mixed ester, polyamide, polypropylene, polyethylene, polycarbonate, polyester and polyimide. Or the method for separating and recovering particles according to 2.
4. The method for separating and recovering particles according to any one of claims 1 to 3, wherein the difference between the maximum value and the minimum value of the pore diameter of the track-etched membrane is within 20% of the maximum value. ..
5. The method for separating and recovering particles according to any one of claims 1 to 4, wherein the particles are extracellular vesicles.
6. The method for separating and recovering particles according to any one of claims 1 to 5, wherein the particles are exosomes.
7. An upstream chamber for receiving a sample solution containing particles with a size of 30 nm to 10 μm, and
   A downstream chamber communicating with the upstream chamber,
   Collect the particles in the sample solution 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. A particle separation and recovery device with a track-etched chamber for use.
8. The particle separation / recovery device according to claim 7, wherein the track-etched membrane has a pore diameter of 10 nm or more and 10 μm or less.
9. 7. The material of the track-etched membrane is any one of polytetrafluoroethylene, polyvinylidene fluoride, polyether sulfone, cellulose mixed ester, polyamide, polypropylene, polyethylene, polycarbonate, polyester and polyimide. Or the particle separation / recovery device according to 8.
10. The particle separation / recovery apparatus according to any one of claims 7 to 9, wherein the difference between the maximum value and the minimum value of the pore diameter of the track-etched membrane is within 20% of the maximum value. ..

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