JP2021010339A - Exosome removal device - Google Patents

Abstract

To provide a device for easily and inexpensively removing exosomes existing in a biological sample, such as serum.SOLUTION: The present invention is a device for removing exosomes from a liquid biological sample. The device is a hollow fiber module type in which a cylindrical container stores a hollow fiber membrane on which a phosphatidylserine binding protein is immobilized. A method for adsorbing and removing exosomes includes bringing a liquid biological sample into contact with the device.SELECTED DRAWING: Figure 1

Description

The present invention relates to a device for efficiently removing exosomes from a biological sample.

Exosomes are vesicle granules having a structure surrounded by a lipid bilayer membrane and are present in all tissues in the living body. Inside the exosome, various proteins such as cytokines and microRNA (miRNA) are contained, and the function of mediating cell-cell communication in the living body and the relationship with diseases such as cancer are attracting attention all over the world. Research is underway. On the other hand, in the field of life science research such as preclinical research on drug discovery and regenerative medicine research, cultured human and animal cells are often used to investigate the effects and effects in vivo. For example, media used in cell culture often require fetal bovine serum (FBS) for cell growth and proliferation, but fetal bovine serum contains large amounts of bovine-derived exosomes. Therefore, since this exosome causes a decrease in reliability in a specific research field, it is required to use fetal bovine serum from which exosome has been removed.

Patent Document 1 describes a carrier and a method for efficiently obtaining exosomes present in a sample by using a protein that specifically binds to a membrane component of exosomes, but further for a large amount of samples. There is a need for a device that can be performed efficiently and easily.

International release WO2016 / 0888689

An object of the present invention is to provide a device for easily and inexpensively removing exosomes existing in a biological sample such as serum.

As a result of diligent studies to solve the above-mentioned problems, the present inventor has found that the above-mentioned problems can be solved by the means shown below, and has arrived at the present invention.

That is, the present invention has the following configuration.
1. 1. A device for removing exosomes from a liquid biological sample, which is a hollow fiber module type device in which a hollow fiber membrane on which a phosphatidylserine-binding protein is immobilized is stored in a container.
2. 2. 2. The device according to 1, wherein the phosphatidylserine binding protein is T-cell antibody-mucin-domain 1 or T-cell antibody-mucin-domain 4.
3. 3. The device according to 1 or 2, wherein the hollow fiber membrane is immobilized with 0.1 mg to 12 mg of phosphatidylserine-binding protein per 1 g of the hollow fiber membrane.
4. The device according to any one of 1 to 3, wherein the hollow fiber membrane has an inner diameter of 20 μm to 1,000 μm.
5. The device according to any one of 1 to 4, wherein the hollow fiber membrane has a film thickness of 10 μm to 300 μm.
6. A method for adsorbing and removing exosomes by bringing a liquid biological sample into contact with the device according to any one of 1 to 5.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a device for easily and inexpensively removing exosomes existing in a biological sample such as serum.

It is a schematic diagram which shows an example of the device of this invention.

The present invention relates to a device for removing exosomes from a liquid biological sample. Specifically, exosomes in a biological sample are removed by contacting a liquid biological sample with a hollow fiber membrane having a phosphatidylserine-binding protein immobilized on the surface.

(Hollow fiber membrane)
In the present invention, the material of the hollow filament membrane constituting the device may be any material as long as it can immobilize the phosphatidylserine-binding protein on the surface of the membrane, and is not particularly limited, but for example, cellulose ester, regenerated cellulose and the like. Cellulosic polymers, polyolefin polymers such as polyethylene and polypropylene, polysulfone polymers such as polysulfone and polyethersulfone, fluoropolymers such as polyvinylidene fluoride (PVDF), polyvinyl alcohol, polymethylmethacrylate, polyacrylonitrile, polyamide, etc. Can be mentioned. Further, these derivatives may be the main component. Further, the hollow fiber membrane used in the present invention may be a material obtained by further modifying these materials. For example, the hollow fiber membrane used may be hydrophilized. By hydrophilizing the hollow fiber membrane, the passage of liquid components inside and outside the membrane becomes easy. Examples of the method for hydrophilizing the hollow fiber membrane include a method for treating the hollow fiber membrane with a hydrophilic polymer such as hydroxyalkyl cellulose or ethylene-vinyl alcohol copolymer, glycerin, or ethanol.

In the present invention, the inner diameter of the hollow fiber membrane is preferably 20 μm to 1000 μm, more preferably 50 μm to 500 μm, and further preferably 100 μm to 500 μm. The film thickness is preferably about 10 μm to 300 μm, more preferably 10 μm to 200 μm, and even more preferably 15 μm to 150 μm. If the inner diameter or film thickness is too large, the device will grow unnecessarily. On the other hand, if the inner diameter is too small, the pressure loss and the linear velocity increase when the liquid is passed through the hollow fiber membrane cavity, which may damage the biological sample. Further, if the film thickness is too small, the strength of the device may not be maintained.

In the present invention, the average pore diameter of the hollow fiber membrane is not particularly limited, and may have pores of a size that allows exosomes to pass through, or a size that does not allow exosomes to pass through but other substances pass through. It may have a hollow fiber. It is more preferable that the exosome has a pore size that allows the exosome to pass through because the adsorption site in the membrane pore can be utilized and the adsorption capacity becomes large. Since the exosome has a diameter of 30 nm to 100 nm, the pore diameter of the hollow fiber membrane is preferably 0.01 μm to 0.7 μm, more preferably 0.1 μm to 0.5 μm.

(Phosphatidylserine binding protein)
In the present invention, the phosphatidylserine-binding protein (protein having a binding ability to phosphatidylserine) is, for example, Annexin V, MFG-E8, Tim1 (T-cell immunoglobulin-mucin-domain 1), Tim3 (T-cell antibody). -Mucin-domain 3), Tim4 (T-cell antibody-mucin-domain 4), etc. have been reported (The Journal of Biochemistry 265, 4923-4928 (25 March 1890), 4923-4928 (25 March 1890), N 2002), Nature 450, 435-439 (15 November 2007). Of these proteins, Tim1, Tim3 and Tim4 are preferably used.

In the present invention, the hollow fiber membrane preferably has phosphatidylserine-binding protein immobilized in an amount of 0.1 mg to 12 mg per 1 g of the hollow fiber membrane. If the amount of phosphatidylserine-binding protein immobilized is small, exosomes may not be sufficiently adsorbed and removed. In addition, if the amount of phosphatidylserine-binding protein immobilized is too large, the manufacturing cost of the device will increase. Therefore, the amount of phosphatidylserine-binding protein immobilized on the hollow fiber membrane is more preferably 0.1 mg / g to 6 mg / g, still more preferably 0.1 mg / g to 3 mg / g.

In the present invention, when exosomes in a biological sample are adsorbed on a hollow fiber membrane on which a phosphatidylserine-binding protein is immobilized, it is preferable that calcium ions (Ca ²⁺ ) coexist in the liquid biological sample. Specifically, it is preferable that the liquid biological sample contains 0.5 mM to 100 mM of Ca ²⁺ , more preferably 0.5 mM to 10 mM, and even more preferably 0.5 mM to 3 mM. When plasma or serum is used as the liquid biological sample, Ca ²⁺ may or may not be added because the serum contains ~ 1.33 mM free Ca ²⁺ .

(Manufacturing of hollow fiber membrane)
In the present invention, the hollow fiber membrane can be produced by a conventionally known method. For example, a film-forming solution obtained by kneading and dissolving a solvent-soluble polymer, a solvent, and if necessary a non-solvent was discharged from a nozzle for molding hollow fibers (three-divided nozzle, double tube nozzle, etc.) and passed through an aerial traveling portion. After that, it is immersed in a coagulation bath to fix the hollow fiber shape. After that, the hollow fiber membrane can be produced by washing and removing excess solvent, non-solvent, and the like. If necessary, the obtained hollow fiber membrane may be dried after attaching a pore-retaining agent.

(Manufacturing of devices)
An appropriate number of dried hollow fiber membranes are bundled and loaded into a plastic case or the like, and the hollow fiber membrane cavity (hollow part) is adhesively fixed to the end of the case with an adhesive resin in a state of being opened. A hollow fiber modular device can be manufactured by attaching a cap having a port communicating with the hollow fiber membrane lumen to the end of the plastic case.

In the present invention, the method for immobilizing the phosphatidylserine-binding protein on the surface (pore surface) of the hollow fiber membrane made of various materials is not particularly limited, and a solution in which the phosphatidylserine-binding protein is dissolved is applied to the hollow fiber membrane lumen and /. Alternatively, a hollow fiber membrane on which the phosphatidylserine-binding protein is immobilized can be obtained by contacting with the outer cavity of the hollow fiber membrane, washing, blocking treatment and / or drying. The phosphatidylserine-binding protein may be immobilized on the membrane surface after the hollow fiber membrane is manufactured, or after the hollow fiber modular device is manufactured. The phosphatidylserine-binding protein may be immobilized only on the inner surface of the hollow fiber membrane, may be immobilized only on the outer surface, or may be immobilized on the inner surface and the outer surface. It may be immobilized on the surface of the membrane and the surface of the pores.

The hollow fiber module (device) manufactured in this manner will be described with reference to FIG. The module case 4 has two ports 2a and 2b that communicate with the outside (outer cavity) of the hollow fiber membrane. The shape of the module case 4 is not particularly limited, but a cylinder or a rectangular parallelepiped is preferable. A plurality of hollow fiber membranes 5 are loaded in such a module case 4, and are fixed by an adhesive resin 6 with the ends of the hollow fiber membranes open. On the other hand, caps 3a and 3b with ports that communicate with the lumen of the hollow fiber membrane are attached to both ends of the module case 4.

As a method for removing exosomes from a liquid biological sample using the hollow fiber modular device of the present invention, for example, a biological sample pressurized in the outer cavity (outer side) or inner cavity (inner side) of the hollow fiber membrane is used. Exosomes can be removed from a biological sample by introducing and filtering from the outside (inside) to the inside (outside) of the hollow fiber membrane and adsorbing exosomes on the membrane surface and / or the pore surface. The higher the removal rate of exosomes from the liquid biological sample, the higher the better, preferably 75% or more, more preferably 80% or more, and even more preferably 85% or more.

(Target sample)
In the present invention, the humoral biological sample is not particularly limited, but includes serum, plasma, urine, cerebrospinal fluid, lymph, ascites, milk, cell culture medium, homogenized extract, and the like. Can be done.

Hereinafter, the effectiveness of the present invention will be described with reference to examples, but the present invention is not limited thereto. A typical implementation method is described below.

[Example 1]
(Preparation of hollow fiber membrane)
Made by uniformly dissolving 20% by mass of polyether sulfone (Ultrason (trade name) E6020P manufactured by BASF), 36% by mass of N-methylpyrrolidone (manufactured by Mitsubishi Chemicals), and 44% by mass of triethylene glycol (manufactured by Mitsui Chemicals). A membrane solution was prepared. This solution was discharged from a cylindrical double tube nozzle heated to 70 ° C. as a hollow forming agent together with a mixed solution of 13.5% by mass of N-methylpyrrolidone, 16.5% by mass of triethylene glycol, and 70% by mass of water. After passing through a 300 mm dry portion, it was immersed in water at 75 ° C. to solidify and wound into a nozzle. A bundle having a length of 20 cm from the wound hollow fiber membrane and 500 hollow fibers was prepared, and then immersed in a 50% glycerin aqueous solution at 50 ° C. to fill the pores with the glycerin aqueous solution. Then, the excess glycerin aqueous solution adhering to the surface was removed, and the mixture was air-dried at 60 ° C. The obtained hollow fiber membrane had an inner diameter of 201 μm, an outer diameter of 301 μm, and a film thickness of 50 μm.

(Making a hollow fiber module)
A hollow fiber module for testing was prepared as follows. After filling 216 hollow fiber membranes in a cylindrical polycarbonate module case with an inner diameter of 0.7 cm and a length of 10 cm, both ends of the module case are filled with a polyurethane-based potting agent so as not to block the hollow part of the hollow fiber membrane. A hollow fiber module having a shape as shown in FIG. 1 was produced.

(Phosphatidylserine binding protein immobilization)
30 mL of a solution (Tim4 solution) prepared by dissolving Tim4 (manufactured by BioLegend) in phosphate buffered saline to a concentration of 20 μg / mL was added to the hollow fiber membrane lumen and outer cavity in the hollow fiber module. Circulated to both. After removing the Tim4 solution from the inside of the hollow fiber module, the hollow fiber membrane lumen and the outer cavity were lightly washed with a phosphate buffered saline to remove the phosphate buffered saline.

(Blocking process)
After the above-mentioned phosphatidylserine-binding protein (Tim4) was immobilized on the hollow fiber membrane, a blocking treatment was performed to prevent non-specific adsorption of the protein or the like. That is, after washing with phosphate buffered saline, both the hollow fiber membrane lumen and the outer cavity of the hollow fiber membrane were filled with a 5% bovine serum albumin solution and allowed to stand in a room temperature environment for 3 hours. The solution was discharged and blocking treatment was performed to prepare a device.

[Example 2]
A device was prepared in the same manner as in Example 1 except that 30 mL of a solution prepared by dissolving Tim4 (manufactured by BioLegend) in phosphate buffered saline at a concentration of 50 μg / mL was used.

[Example 3]
(Preparation of hollow fiber membrane)
Cellulose triacetate (Daicel Chemicals) 17% by mass, N-methylpyrrolidone (Mitsubishi Chemicals) 58.1% by mass and triethylene glycol (Mitsui Chemicals) 24.9% by mass are uniformly dissolved to prepare a film-forming solution. did. The obtained film-forming solution was discharged from a cylindrical double-tube nozzle together with water as a hollow forming agent, passed through a 30 mm dry portion, and then N-methylpyrrolidone was 54.6% by mass and triethylene glycol was 23.4% by mass. , Was introduced into a coagulation liquid of 40 ° C. consisting of 22% by mass of water and solidified. The obtained hollow fiber membrane was washed with water, treated with glycerin, and then dried and wound. The obtained hollow fiber membrane had an inner diameter of 250 μm, an outer diameter of 308 μm, and a film thickness of 29 μm.
A device was produced in the same manner as in Example 1 using the 200 hollow fiber membranes obtained.

[Example 4]
(Preparation of hollow fiber membrane)
A membrane-forming solution is prepared by uniformly dissolving 19% by mass of cellulose triacetate (Dycel Chemical Industries, Ltd.), 56.7% by mass of N-methylpyrrolidone (Mitsubishi Chemicals, Inc.) and 24.3% by mass of triethylene glycol (Mitsui Chemicals, Inc.). did. The obtained film-forming solution was discharged from a cylindrical double-tube nozzle together with liquid paraffin as a hollow forming agent, passed through a 5 mm dry portion, and then N-methylpyrrolidone 14% by mass, triethylene glycol 6% by mass, and water 80. It was solidified by being guided into a coagulation liquid of 40 ° C. consisting of mass%. The obtained hollow fiber membrane was washed with water, subjected to glycerin adhesion treatment, dried and wound up. The obtained hollow fiber membrane had an inner diameter of 400 μm, an outer diameter of 600 μm, and a film thickness of 100 μm.
A device was produced in the same manner as in Example 1 using the 128 hollow fiber membranes obtained. The module case used had an inner diameter of 1.1 cm.

[Example 5]
(Preparation of hollow fiber membrane)
A membrane-forming solution is prepared by uniformly dissolving 19% by mass of cellulose triacetate (Dycel Chemical Industries, Ltd.), 56.7% by mass of N-methylpyrrolidone (Mitsubishi Chemicals, Inc.) and 24.3% by mass of triethylene glycol (Mitsui Chemicals, Inc.). did. The obtained film-forming solution was discharged from a cylindrical double-tube nozzle together with liquid paraffin as a hollow forming agent, passed through a 5 mm dry portion, and then N-methylpyrrolidone 14% by mass, triethylene glycol 6% by mass, and water 80. It was solidified by being guided into a coagulation liquid of 40 ° C. consisting of mass%. The obtained hollow fiber membrane was washed with water, subjected to glycerin adhesion treatment, dried and wound up. The obtained hollow fiber membrane had an inner diameter of 150 μm, an outer diameter of 184 μm, and a film thickness of 17 μm.
A device was produced in the same manner as in Example 1 using the obtained 600 hollow fiber membranes.

[Example 6]
Polyether sulfone (Sumika Excel (registered trademark) 4800P manufactured by Sumitomo Chemtech) 19.0% by mass, polyvinylpyrrolidone manufactured by BASF (registered trademark K30) 3.0% by mass, N-methylpyrrolidone (manufactured by Mitsubishi Chemicals) A film-forming solution was prepared by uniformly dissolving 35.1% by weight and 42.9% by weight of triethylene glycol (manufactured by Mitsui Chemicals, Inc.). The obtained film-forming solution is discharged from a cylindrical double-tube nozzle at 72 ° C. together with a mixed solution of 35.1% by mass of N-methylpyrrolidone, 42.9% by mass of triethylene glycol, and 22% by mass of RO water as a hollow forming agent. Then, after passing through a 20 mm dry portion, it was immersed in a mixed solution of 13.5% by mass of N-methylpyrrolidone, 16.5% by mass of triethylene glycol, and 70.0% by mass of RO water to coagulate and wind up in a skein. A bundle having a length of 20 cm and 428 hollow fibers was prepared from the wound hollow fiber membrane, and then air-dried at 60 ° C. The obtained hollow fiber membrane had an inner diameter of 200 μm, an outer diameter of 300 μm, and a film thickness of 50 μm.
A device was produced in the same manner as in Example 1 using the obtained hollow fiber membrane. In addition, Tim1 was used as a phosphatidylserine binding protein.

[Comparative Example 1]
Phosphatidylserine binding protein is bound to streptavidin magnetic beads included in a magnetic bead type exosome purification kit (MagCaptureTM Exosome Isosome Isolation Kit PS, manufactured by Wako Junyaku) according to the procedure described in the instruction manual of the product. Serin-binding protein-immobilized magnetic beads were prepared. 60 mg of these beads were added to 1 mL of serum placed in a 1.5 mL tube, and the beads were inverted and mixed in an environment of 4 ° C. for 3 hours to bind serum exosomes.

[Comparative Example 2]
A device was prepared in the same manner as in Example 1 except that the phosphatidylserine binding protein was not immobilized.

[Comparative Example 3]
A device was prepared in the same manner as in Example 3 except that the phosphatidylserine binding protein was not immobilized.

(Exosome adsorption test)
For each device of Examples 1 to 6 and Comparative Examples 2 and 3, 20 mL of human serum is introduced from the port 3a of each device, and the serum discharged from 3b is introduced into 2b and discharged from 2a. The serum discharged from 2a is returned to the original solution and circulated to the device again. This was carried out in an environment of 4 ° C. for 12 hours. In addition, CaCl ₂ was added to human serum so as to have a final concentration of 2 mM.

(Measurement of exosome number)
The number of exosomes in serum before and after the exosome adsorption test was measured in Examples and Comparative Examples using NanoSight LM10 (Malvern Panalytic).

(Measurement of exosome removal rate)
The exosome removal rate was calculated by the following formula using the number of exosomes in the serum collected after the completion of exosome removal and the number of exosomes before removal.
Exosome removal rate (%) = (1-Number of exosomes per 1 mL after removal / Number of exosomes per 1 mL before removal) x 100

For Examples 1-6 and Comparative Example 1-3, the exosome adsorption test was performed 5 times each, and the exosome removal rate was measured. The results are summarized in Table 1.

INDUSTRIAL APPLICABILITY According to the present invention, exosomes existing in a biological sample such as serum can be easily and inexpensively removed, so that analysis and the like excluding the influence of exosomes can be performed.

1 device (hollow fiber module)
2a, 2b Port 3a, Cap with 3b Port 4 Module Case 5 Hollow Fiber Membrane 6 Adhesive Resin

Claims (6)

A device for removing exosomes from a liquid biological sample, which is a hollow fiber module type device in which a hollow fiber membrane on which a phosphatidylserine-binding protein is immobilized is stored in a container. The device according to claim 1, wherein the phosphatidylserine binding protein is T-cell immunoglobulin-mucin-domain 1 or T-cell immunoglobulin-mucin-domain 4. The device according to claim 1 or 2, wherein the hollow fiber membrane is immobilized with 0.1 mg to 12 mg of phosphatidylserine-binding protein per 1 g of the hollow fiber membrane. The device according to any one of claims 1 to 3, wherein the hollow fiber membrane has an inner diameter of 20 μm to 1,000 μm. The device according to any one of claims 1 to 4, wherein the hollow fiber membrane has a film thickness of 10 μm to 300 μm. A method for adsorbing and removing exosomes by bringing a liquid biological sample into contact with the device according to any one of claims 1 to 5.