WO2019168184A1 - Extracellular-vesicle population and method for manufacturing same - Google Patents

Extracellular-vesicle population and method for manufacturing same Download PDF

Info

Publication number
WO2019168184A1
WO2019168184A1 PCT/JP2019/008235 JP2019008235W WO2019168184A1 WO 2019168184 A1 WO2019168184 A1 WO 2019168184A1 JP 2019008235 W JP2019008235 W JP 2019008235W WO 2019168184 A1 WO2019168184 A1 WO 2019168184A1
Authority
WO
WIPO (PCT)
Prior art keywords
extracellular vesicles
population
cells
extracellular
zeta potential
Prior art date
Application number
PCT/JP2019/008235
Other languages
French (fr)
Japanese (ja)
Inventor
一木　隆範
Original Assignee
公益財団法人川崎市産業振興財団
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 公益財団法人川崎市産業振興財団 filed Critical 公益財団法人川崎市産業振興財団
Priority to JP2020503662A priority Critical patent/JP7296937B2/en
Publication of WO2019168184A1 publication Critical patent/WO2019168184A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q90/00—Cosmetics or similar toiletry preparations for specific uses not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions

Definitions

  • the present invention relates to a population of extracellular vesicles and a method for producing the same. Furthermore, the present invention relates to a method for evaluating the quality of a population of extracellular vesicles, a composition containing extracellular vesicles, and the like.
  • Exosome is a kind of extracellular vesicle secreted by cells. Exosomes are attracting attention as candidate disease markers that can be used for early detection of intractable diseases including cancer and determination of therapeutic effects. Exosomes are also expected to be used as carriers for drug delivery systems (DDS) and therapeutic applications such as regenerative medicine using mesenchymal stem cell-derived exosomes.
  • DDS drug delivery systems
  • therapeutic applications such as regenerative medicine using mesenchymal stem cell-derived exosomes.
  • it is difficult to analyze and identify a heterogeneous particle population having a diameter of several tens to 100 nm and a heterogeneity.
  • the present invention has been made in view of the above circumstances, and a method for producing such extracellular vesicles, such as a group of extracellular vesicles of uniform quality and a composition containing such extracellular vesicles, and cells It is an object to provide a method for evaluating the quality of outer vesicles.
  • the present invention includes the following aspects.
  • [1] A population of extracellular vesicles having a standard deviation of zeta potential of extracellular vesicles of 5 mV or less.
  • [2] The population of extracellular vesicles according to [1], wherein the standard deviation of the zeta potential is 4.5 mV or less.
  • [3] The population of extracellular vesicles according to [2], wherein the standard deviation of the zeta potential is 4 mV or less.
  • [4] The population of extracellular vesicles according to [3], wherein the standard deviation of the zeta potential is 3.5 mV or less.
  • [5] The population of extracellular vesicles according to [4], wherein the standard deviation of the zeta potential is 3 mV or less.
  • [6] The population of extracellular vesicles according to any one of [1] to [5], wherein the extracellular vesicle is an exosome.
  • [7] (a) A step of synchronizing the cell cycle of a plurality of cells; (b) After the step (a), the medium of the plurality of cells is replaced with a medium substantially free of extracellular vesicles.
  • Process (C) culturing the plurality of cells in the medium exchanged, (d) recovering a population of extracellular vesicles from the medium after step (c);
  • a method for producing a population of extracellular vesicles comprising: [8] The cell according to [7], wherein the population of extracellular vesicles obtained after the step (d) is the population of extracellular vesicles according to any one of [1] to [6].
  • a method for producing a population of outer vesicles [9] The method for producing a population of extracellular vesicles according to [7] or [8], wherein the step (a) is performed by culturing the plurality of cells in a medium containing a cell cycle synchronizer.
  • [10] The method for producing a population of extracellular vesicles according to [7] or [8], wherein the step (a) is performed by culturing the plurality of cells in a confluent state.
  • the method further includes (e) a step of measuring the zeta potential of the extracellular vesicles contained in the collected extracellular vesicle population.
  • [7] to [11] A method for producing a population of extracellular vesicles according to any one of the above.
  • [13] A population of extracellular vesicles produced by the method for producing a population of extracellular vesicles according to any one of [7] to [12].
  • [14] A method for evaluating the quality of a population of extracellular vesicles, wherein (a) measuring the zeta potential of a plurality of extracellular vesicles contained in the population of extracellular vesicles; Calculating the standard deviation of the zeta potential measured in step (a), and (c) evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b).
  • step (c) when the standard deviation is 5 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality.
  • step (c) when the standard deviation is 4.5 mV or less, it is determined that the uniformity of the population of the extracellular vesicles is high. A method of assessing the quality of a population.
  • step (c) when the standard deviation is 4 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality.
  • step (c) when the standard deviation is 3.5 mV or less, it is determined that the uniformity of the population of extracellular vesicles is high. A method of assessing the quality of a population. [19] In the step (c), when the standard deviation is 3 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality. [20] A composition comprising a plurality of extracellular vesicles, wherein the standard deviation of the zeta potential of the extracellular vesicles contained in the composition is 5 mV or less. [21] The composition according to [20], wherein the standard deviation of the zeta potential is 4.5 mV or less.
  • a pharmaceutical composition comprising the population of extracellular vesicles according to any one of [1] to [6].
  • a cosmetic comprising the population of extracellular vesicles according to any one of [1] to [6].
  • a food comprising the population of extracellular vesicles according to any one of [1] to [6].
  • the food according to [30] which is a health food or a functional food
  • a method for producing an extracellular vesicle and a method for evaluating the quality of the extracellular vesicle such as a group of extracellular vesicles having a uniform quality and a composition containing the extracellular vesicle.
  • FIG. 3 is a partial cross-sectional view of a fluid device partially cut along a yz plane.
  • FIG. 7 is a cross-sectional view taken along line AA in FIG. It is a figure which shows schematic structure of the irradiation part and adjustment part of a fluid device. It is a partial detail drawing of the adjustment part of a fluid device, and a fluid device. It is a figure which shows typically the optical path through which illumination light passes the end surface of a reservoir member, and the side surface of a flow path. It is a figure which shows schematic structure of the control apparatus of a fluid device. It is a figure which shows an example of the particle
  • FIG. 2 is a graph showing the measurement results of the particle size and zeta potential of extracellular vesicles collected in Example 1.
  • the present invention provides a population of extracellular vesicles wherein the standard deviation of the zeta potential of the extracellular vesicle is 5 mV or less.
  • Extracellular vesicles are vesicles released by cells.
  • the size of the extracellular vesicle is about 30 nm to 1 ⁇ m in diameter.
  • An extracellular vesicle is a secreted product of a cell and expresses a cell-derived protein as a secretory source on its surface.
  • Examples of extracellular vesicles include exosomes, apoptotic bodies, microvesicles and the like.
  • a typical example of an extracellular vesicle is exosome. Exosomes are lipid vesicles with a diameter of about 30 to 200 nm.
  • various cells such as tumor cells, dendritic cells, T cells, and B cells can be used for blood, urine, saliva, etc. Secreted into body fluids.
  • Abnormal cells such as cancer cells existing in the body express a protein specific to the cell membrane.
  • An exosome is a secreted product of a cell and expresses a cell-derived protein as a secretory source on its surface.
  • the surface of the exosome is a membrane surface of a lipid vesicle secreted from a cell, and refers to a portion where the secreted exosome is in contact with the environment in the living body.
  • Extracellular vesicles may be processed extracellular vesicles released by cells.
  • the method for processing the extracellular vesicle is not particularly limited as long as the processed extracellular vesicle maintains the vesicle structure.
  • Examples of extracellular vesicle processing methods include modification of the membrane surface of the extracellular vesicle (for example, modification with a peptide, sugar chain, etc.), encapsulation of a drug in the extracellular vesicle, and the like.
  • a group of extracellular vesicles refers to two or more extracellular vesicles, for example, 10 3 or more, 10 4 or more, 10 5 or more, 10 6 or more extracellular vesicles Is done.
  • the upper limit of the number of extracellular vesicles contained in the population of extracellular vesicles is not particularly limited. For example, 10 15 or less, 10 14 or less, 10 13 or less, 10 12 or less, 10 11 or less, Or 10 10 or less are illustrated.
  • the population of extracellular vesicles may be, for example, 10 3 to 10 15 , 10 4 to 10 12 or more, or 10 5 to 10 10 extracellular vesicles.
  • the population of extracellular vesicles of this embodiment is preferably a population of extracellular vesicles obtained by culturing the same type of cells. That is, it is preferable that all the extracellular vesicles constituting the population of extracellular vesicles of this embodiment are extracellular vesicles released from the same type of cells.
  • the cells are not particularly limited as long as they have the ability to release extracellular vesicles. Examples of such cells include various disease cells such as tumor cells; immune cells such as dendritic cells, T cells, and B cells; various tissue cells such as nerve cells; fat cells; mesenchymal stem cells, hematopoietic stem cells, and the like.
  • Somatic stem cells pluripotent stem cells such as ES cells and iPS cells; germ cells and the like, but not limited thereto.
  • the biological species from which the cells are derived is not particularly limited.
  • the cell may be a human cell.
  • cells other than mammals other than humans for example, mice, guinea pigs, monkeys, dogs, cats, cows, horses, pigs, etc. may be used.
  • the extracellular vesicle population includes, for example, various disease cells such as tumor cells; immune cells such as dendritic cells, T cells and B cells; various tissue cells such as nerve cells; adipocytes; mesenchymal stem cells and hematopoietic stem cells.
  • Pluripotent stem cells such as somatic stem cells such as ES cells, iPS cells, etc .; a population of extracellular vesicles released from various cells such as germ cells.
  • the population of extracellular vesicles of this embodiment has a standard deviation of the zeta potential of the extracellular vesicles of 5 mV or less.
  • the standard deviation of the zeta potential is preferably 4.5 mV or less, more preferably 4 mV or less, further preferably 3.5 mV or less, and particularly preferably 3 mV or less.
  • the standard deviation of the zeta potential can be obtained by measuring the zeta potential of individual extracellular vesicles constituting the population of extracellular vesicles and calculating the standard deviation from the measured values.
  • the number of extracellular vesicles for measuring the zeta potential is sufficient if it is sufficient to calculate the standard deviation, and is appropriately selected according to the size of the extracellular vesicle population (number of extracellular vesicles). That's fine.
  • the number of extracellular vesicles for measuring zeta potential can be 100 or more, 300 or more, 500 or more, 600 or more, 700 or more, or 800 or more.
  • the standard deviation of the zeta potential of a population of extracellular vesicles can be calculated by the following formula (s).
  • the population of extracellular vesicles of this embodiment can be produced by a method for producing a population of extracellular vesicles described later.
  • the population of extracellular vesicles of this embodiment is a population of extracellular vesicles having a standard deviation of zeta potential of 5 mV or less and uniform quality. Therefore, it can be used for various uses such as pharmaceuticals, cosmetics, and foods. Since the population of extracellular vesicles of this embodiment has uniform quality, it can be suitably used as a drug delivery system (DDS) carrier or a pharmaceutical product such as regenerative medicine.
  • DDS drug delivery system
  • the zeta potential of individual extracellular vesicles constituting the extracellular vesicle population can be measured by a known method, apparatus, or system. Examples of such a method, apparatus, or system include the method, apparatus, or system described in International Publication No. 2016/171198, International Publication No. 2016/063912, International Publication No. 2014/030590, and the like. Is done. Below, an example of the zeta potential measuring device of an extracellular vesicle is described.
  • the zeta potential is measured by binding a specific binding substance to the extracellular vesicle, but the zeta potential of the extracellular vesicle is measured without binding the specific binding substance. Is also possible.
  • the zeta potential of the extracellular vesicle population of this embodiment is preferably measured without binding a specific binding substance to the extracellular vesicle. That is, in the explanation illustrated below, it is preferable not to perform the binding reaction between the specific binding substance and the extracellular vesicle.
  • the zeta potential of the extracellular vesicle may be measured using a specific binding substance for the protein.
  • the zeta potential of the extracellular vesicle is measured.
  • a population of extracellular vesicles having a standard deviation of zeta potential measured by this method of 5 mV or less is a population of extracellular vesicles having high uniformity with respect to the expression level of a specific membrane protein.
  • the zeta potential of the extracellular vesicle may be measured by binding a specific binding substance specific to each membrane protein to a plurality of types of membrane proteins.
  • a population of extracellular vesicles having a standard deviation of zeta potential measured by this method of 5 mV or less is a population of extracellular vesicles with high uniformity in the expression levels of the plurality of types of membrane proteins.
  • extracellular vesicle zeta potential refers to the zeta potential measured without binding a specific binding substance to the extracellular vesicle, specific to a specific membrane protein in the extracellular vesicle.
  • zeta potential specific binding substance-data potential of extracellular vesicle complex
  • Zeta potentials data potentials of multiple specific binding substances-extracellular vesicle complexes.
  • the plurality of types of membrane proteins are not particularly limited as long as they are two or more types, and examples thereof include 2 to 50 types, 2 to 30 types, 2 to 20 types, and 2 to 10 types.
  • the “specific binding substance” means a substance having an ability to specifically bind to a specific molecule (for example, a protein).
  • the phrase “specifically binds to a specific molecule” means that it has a high binding affinity for the specific molecule and a low binding affinity for other molecules.
  • the specific binding substance varies depending on the specific molecule to be bound, and can be variously selected according to the type of the specific molecule.
  • Specific molecules to be bound by specific binding substances include molecules present on the surface of extracellular vesicles, and examples include antigens, membrane proteins, nucleic acids, sugar chains, glycolipids, and the like.
  • Specific binding substances for proteins include, for example, antibodies (including chimeric antibodies, humanized antibodies, modified antibodies, multivalent antibodies, multispecific antibodies, modified antibodies such as antibody fragments), aptamers (nucleic acid aptamers, peptide aptamers, etc.) And ligand molecules.
  • the class of the antibody as a specific binding substance is not particularly limited, and may be any antibody class such as IgG, IgA, IgD, IgE, and IgM.
  • Examples of IgG include IgG1, IgG2, IgG3, and IgG4.
  • Examples of IgA include IgA1 and IgA2.
  • IgM include IgM1 and IgM2.
  • Examples of antibody fragments include scFv, Fab, F (ab ') 2, Fv and the like.
  • the ligand molecule include a ligand of the receptor protein when the specific molecule to be bound is a receptor protein.
  • examples of the ligand molecule include G protein.
  • the specific binding substance may be labeled with a labeling substance.
  • labeling substances include biotin, avidin, streptavidin, neutravidin, glutathione-S-transferase, glutathione, fluorescent dyes, polyethylene glycol, charged molecules such as melittic acid, and the like.
  • FIG. 1 is a schematic plan view of a particle detection apparatus 100 that can be used for zeta potential measurement.
  • FIG. 2 is a schematic front view of the particle detection apparatus 100.
  • the particle detection apparatus 100 detects information related to particles in the fluid device C by irradiating the fluid device C with the illumination light L1 and observing the scattered light L2 from the fluid device C with the fluid device C as a detection target.
  • the particle detection apparatus 100 includes a light source unit LS, an irradiation unit 20, an adjustment unit CL, a stage unit ST, a detection unit 30, a transmission unit 40, and a control device 5.
  • a particle detection system 1 is configured by the particle detection apparatus 100 and the fluid device C.
  • the direction orthogonal to the orthogonal surface (not shown) orthogonal to the installation surface STa of the stage part ST is the x direction (x axis; third direction), and the direction parallel to the installation surface STa and orthogonal to the x direction.
  • the y direction (y axis) and the vertical direction perpendicular to the x direction and the y direction are appropriately described as the z direction (z axis; second direction).
  • the fluid device C that is a detection target will be described.
  • the fluid device C in this embodiment is an electrophoresis analysis chip used when analyzing an extracellular vesicle as an example.
  • an extracellular vesicle analysis chip (electrophoresis analysis chip) will be described by taking an example of analyzing exosomes as extracellular vesicles.
  • analysis of exosomes using an extracellular vesicle analysis chip can be performed as follows. First, the exosome to be detected is purified. Next, the exosome is brought into contact with the specific binding substance. As the specific binding substance, a substance that can specifically bind to a molecule present on the surface of the exosome is selected. Next, the zeta potential of exosome is measured and analyzed using an extracellular vesicle analysis chip. This analysis can be applied not only to exosomes but also to analysis of extracellular vesicles in general. In addition, the exosome may be subjected to analysis without contact with a specific binding substance.
  • exosomes are purified from a sample containing exosomes.
  • the sample include a cell culture solution.
  • Examples of the method for purifying exosomes include ultracentrifugation, ultrafiltration, continuous flow electrophoresis, chromatography, and a method using a ⁇ -TAS (Micro-Total Analysis Systems) device.
  • ⁇ -TAS Micro-Total Analysis Systems
  • the exosome is brought into contact with the specific binding substance.
  • a specific binding substance-exosome complex is formed.
  • an abnormality associated with a disease such as cancer, obesity, diabetes, or neurodegenerative disease can be detected.
  • the expression level of the molecule to be detected on the exosome surface can be analyzed. For example, it is possible to evaluate exosomes with altered functions, such as using a specific binding substance that specifically binds to the peptide or protein of the exosome that artificially expresses the peptide or protein on the membrane surface. it can.
  • the zeta potential of the exosome reacted with the antibody is measured.
  • the zeta potential is the surface charge of the fine particles in the solution.
  • exosomes are negatively charged while antibodies are positively charged.
  • the zeta potential of the antibody-exosome complex is shifted positively compared to the zeta potential of the exosome alone. Therefore, by measuring the zeta potential of the exosome reacted with the antibody, the expression of the antigen on the exosome membrane surface can be detected. This is true not only for antibodies but also for other positively charged specific binding substances.
  • the exosome zeta potential ⁇ is obtained by performing exosome electrophoresis in the microchannel of an extracellular vesicle analysis chip, optically measuring the exosome electrophoresis speed S, and measuring the measured exosome electrophoresis. Based on the speed S, it can be calculated using the Smolkovsky equation shown in the following equation (1).
  • Equation (1) U is the electrophoretic mobility of the exosome to be measured, and ⁇ and ⁇ are the dielectric constant and viscosity coefficient of the sample solution, respectively.
  • the electrophoretic mobility U can be calculated by dividing the electrophoretic velocity S by the electric field strength in the microchannel.
  • the exosome electrophoresis speed S is obtained by electrophoresing an exosome in a microchannel of an extracellular vesicle analysis chip, and as an example, irradiating the exosome flowing in the microchannel with a Rayleigh Measurement can be performed by obtaining a particle image by scattered light.
  • the laser beam one having a wavelength of 405 nm and an intensity of 150 mW can be given.
  • the particle diameter d of the exosome is obtained by performing exosome electrophoresis in the microchannel of the extracellular vesicle analysis chip, optically measuring the exosome electrophoresis speed S, and measuring the exosome electrophoresis. Based on the speed S, it can be calculated using the Einstein-Stokes equation shown in the following equation (2).
  • d is the particle diameter of exosome
  • k is Boltzmann constant
  • T absolute temperature
  • ⁇ is the viscosity coefficient of the sample solution
  • D is the diffusion coefficient of the fine particles. That is, the particle diameter d of the exosome can be calculated based on the Brownian motion state of the exosome to be measured.
  • FIG. 3 is a perspective view showing the basic structure of the extracellular vesicle analysis chip.
  • 4 is a cross-sectional view taken along line II-II in FIG.
  • the extracellular vesicle analysis chip CH includes a first reservoir 110, a second reservoir 120, an electrophoresis channel 150 that connects the first reservoir 110 and the second reservoir 120, and a base material 160.
  • the migration channel 150 is, for example, a millimeter channel or a micro channel.
  • the migration channel 150 has a width of about 200 ⁇ m, a height of 400 ⁇ m, and a length of about 10 mm.
  • the electrophoresis channel 150 is a specific binding formed by interaction between an extracellular vesicle or a specific binding substance that specifically binds to a biomolecule existing on the surface of the extracellular vesicle and the extracellular vesicle.
  • the substance-extracellular vesicle complex (for example, antibody-exosome complex) is electrophoresed.
  • the electrophoresis channel 150 has one end connected to the first reservoir 110 and the other end connected to the second reservoir 120.
  • the first reservoir 110 and the second reservoir 120 are provided on the base material 160 and have an electrode 130 and an electrode 140, respectively.
  • the electrode 130 is provided at the bottom of the first reservoir 110
  • the electrode 140 is provided at the bottom of the second reservoir 120.
  • the electrode 130 and the electrode 140 are each provided in the vicinity of the end of the migration channel 150.
  • a sample eg, exosome to be analyzed
  • a buffer solution is introduced into the second reservoir 120. Note that the buffer solution may be introduced into the first reservoir 110.
  • the extracellular vesicle analysis chip CH is suitable for measuring the zeta potential of extracellular vesicles.
  • a method for measuring the zeta potential of exosomes using the extracellular vesicle analysis chip CH will be described, taking as an example the case of analyzing exosomes as extracellular vesicles.
  • a sample solution containing exosomes to be analyzed is introduced into the first reservoir 110.
  • the exosome to be analyzed may have been reacted with a specific binding substance.
  • the exosome is obtained from, for example, the culture supernatant, and the sample solution is an exosome suspension in which the exosome is suspended in a buffer solution such as a phosphate buffer (Phosphate Buffered Saline, PBS).
  • a sample solution containing exosomes is introduced into the migration channel 150.
  • the exosome can be introduced into the electrophoresis channel 150 by connecting a syringe to the second reservoir 120 and sucking the sample solution.
  • the buffer solution is put into the first reservoir 110 and the second reservoir 120.
  • liquid level (liquid level height) between the first reservoir 110 and the second reservoir 120 By adjusting the liquid level (liquid level height) between the first reservoir 110 and the second reservoir 120 by using a liquid level adjusting means, which will be described later, it is possible to prevent the generation of a hydrostatic pressure flow that occurs in the migration channel 150 and to measure the zeta potential. The accuracy can be improved.
  • a voltage is applied between the electrodes 130 and 140 by a control unit (for example, a control device 5 described later or a computer), and the exosome is electrophoresed.
  • the control unit applies a voltage having an electric field strength of about 50 V / cm for about 10 seconds.
  • the electrophoresis channel 150 is irradiated with laser light, and the scattered light passing through the exosome, which is emitted from the electrophoresis channel 150, is collected using an objective lens or the like, and a light receiving sensor (eg, high A sensitivity camera is used to image exosomes or specific binding substance-exosome complexes.
  • the magnification of the objective lens is about 60 times as an example.
  • the wavelength and intensity of the laser are, for example, a wavelength of 405 nm and an intensity of 150 mW.
  • the extracellular vesicle analysis chip CH By using the extracellular vesicle analysis chip CH, not only the average value of the zeta potential of the exosome or specific binding substance-exosome complex, but also the zeta potential of the exosome or specific binding substance-exosome complex at the level of one particle. It can be measured. Therefore, the standard deviation of the zeta potential in a population of exosomes can be determined from the zeta potential of individual exosomes or specific binding substance-exosome complexes.
  • the exosome or the specific binding substance-exosome complex may be simply referred to as “exosome”.
  • FIG. 5 is a plan view in which the fluid device C is installed on the installation surface STa of the stage unit ST.
  • FIG. 6 is a partial cross-sectional view in which the fluid device C is partially cut in the yz plane.
  • 7 is a cross-sectional view taken along line AA in FIG.
  • the fluid device C is formed in a rectangular shape in plan view.
  • the fluid device C includes a reservoir member (first base material) 10 and a bottom plate (second base material) 11 that are sequentially stacked in the z direction.
  • the fluid device C has a laminated structure (laminated body) composed of at least the reservoir member 10 and the bottom plate 11.
  • the laminated structure of the fluid device C has a two-layer structure.
  • such a laminated structure of the fluid device C is formed by bonding the reservoir member 10 and the bottom plate 11 to each other.
  • the reservoir member 10 is formed of a material that can be elastically deformed in at least one direction by an external force or the like.
  • Examples of the material of the reservoir member 10 include elastomers such as silicone rubber and PDMS (polydimethylsiloxane).
  • the bottom plate 12 is made of a material through which scattered light L2 generated by irradiation with the illumination light L1 is transmitted.
  • the bottom plate 12 is formed of a glass material as an example.
  • the fluidic device C includes a plurality of (three in FIG. 5) lanes 2 arranged in the length direction (y direction).
  • Each lane 2 includes a first reservoir 12A, a second reservoir 12B, a flow path 13, and electrodes 18A and 18B.
  • the first reservoir 12A and the second reservoir 12B are arranged at an interval in the y direction.
  • the first reservoir 12 ⁇ / b> A and the second reservoir 12 ⁇ / b> B are arranged at an interval in the flow path direction of the flow path 13.
  • the plurality of lanes are arranged in the flow path direction (in series), so that it is easy to irradiate light from the side.
  • a plurality of lanes may be analyzed in order for each lane, or may be analyzed simultaneously by a plurality of detection systems.
  • the plurality of lanes 2 may be arranged in the height direction (z direction).
  • the solution may be injected from the length direction (x direction) or from the y direction.
  • there are a plurality of irradiation light sources and each of the light sources irradiates fine particles flowing through the lane 2 having a corresponding height. Further, the fine particles flowing in the lane 2 may be irradiated by changing the irradiation direction from at least one irradiation light source.
  • the illumination light applied to each lane 2 may be adjusted by adjusting the shape of the illumination light by moving the objective lens. Further, when there are a plurality of lanes 2, a configuration in which the measurement target lane 2 is selected (switched) from among the plurality of lanes 2 by moving a stage on which the fluid device C is placed may be employed.
  • the first reservoir 12A has a holding space 14A that has a circular cross section in a plane parallel to the xy plane and extends in the z direction, and a funnel shape that gradually increases in diameter from the + z side end of the holding space 14A toward the + z direction.
  • the introduction part 15A is provided.
  • the holding space 14A opens at the ⁇ z side end facing the bottom plate 11.
  • the holding space 14 ⁇ / b> A is connected to the flow path 13.
  • the second reservoir 12B has a holding space 14B having a circular cross section in a plane parallel to the xy plane and extending in the z direction, and a funnel shape gradually increasing in diameter from the + z side end of the holding space 14B toward the + z direction.
  • the introduction part 15B is provided.
  • the holding space 14B has an end on the ⁇ z side facing the bottom plate 11 and opening.
  • the holding space 14 ⁇ / b> B is connected to the flow path 13.
  • the flow path 13 is a flow path for electrophoresis (flow path for electrophoresis).
  • the flow path 13 extends in the y direction, which is the length direction of the fluid device C.
  • the channel 13 is provided on the surface facing the bottom plate 11 so as to connect the holding space 14A and the holding space 14B.
  • the flow path 13 is formed in a rectangular cross section surrounded by the groove 10 ⁇ / b> A formed in the reservoir member 10 and the surface (second surface) 11 a of the bottom plate 11.
  • the groove portion 10A is formed to be surrounded by side surfaces (first surfaces) 16a and 16b facing in the x direction and a bottom surface (second surface) 16c facing the surface 11a of the bottom plate 11 in the z direction.
  • the side surfaces 16a, 16b, the bottom surface 16c, and the surface 11a constituting the groove 10A are mirror-finished.
  • the first surface includes a side surface 16a that is a first side surface and a side surface 16b that is a second side surface.
  • the side surface 16a and the side surface 16b face each other and are separated from each other in the x direction which is the first direction.
  • the lane 2 is arranged so as to be biased toward the side closer to the end surface 17 on the + x side than the center with respect to the optical axis direction (incident direction) of the illumination light L1 that is the width direction of the fluid device C.
  • the lane 2 is arranged so as to be biased toward the side closer to the end surface 17 on the incident side of the illumination light L1 than the center in the width direction (the x direction in FIG. 5) of the fluid device C that is the optical axis direction of the incident illumination light L1.
  • the end face 17 is mirror-finished in a range where at least the lane 2 is provided in the y direction.
  • the channel 13 is formed in a size of about 200 ⁇ m in width, about 400 ⁇ m in height (depth of the groove 10A), and about 10 mm in length.
  • An electrode 18A is provided on the surface 11a of the bottom plate 11 so as to face the holding space 14A.
  • An electrode 18B is provided on the surface 11a of the bottom plate 11 so as to face the holding space 14B.
  • Examples of the material for the electrode 18A and the electrode 18B include gold, platinum, and carbon.
  • the end surface (second end surface) 19 located on the incident side of the illumination light L1 in the bottom plate 11 is closer to the incident side of the illumination light L1 than the position of the end surface 17 of the reservoir member 10 in the x direction. Are spaced apart on the opposite side, -x side.
  • the light source unit LS has a wavelength that does not adversely affect the particles.
  • the light source unit LS has a wavelength of 405 nm and an intensity of 150 mW.
  • Laser light having a deflection direction in the z direction at 0.8 mm is emitted as illumination light L1.
  • the illumination light L1 may be polarized light (for example, linearly polarized light) or non-polarized light. However, in the present embodiment, vertical polarization is used and there is no directivity of Rayleigh scattering.
  • the illumination light L1 is applied to the fluid device C along an optical axis extending in a direction intersecting the orthogonal plane described above. In the present embodiment, the optical axis of the illumination light L1 is parallel to the x direction.
  • the illumination light L1 of the present embodiment is applied to the fluid device C along the optical axis extending in the x direction.
  • FIG. 8 is a diagram illustrating a schematic configuration of the irradiation unit 20 and the adjustment unit CL.
  • the irradiation unit 20 includes a ⁇ / 2 plate 21 and an expander lens 22 that are sequentially arranged along the optical axis of the illumination light L1.
  • the optical axis of the illumination light L1 extends in the y direction, but the illumination light L1 that finally irradiates the fluid device C (channel 13) is x. Since the optical axis is along the direction, the illumination light L1 shown in FIG. 8 is illustrated on the assumption that the optical axis is along the x direction.
  • the illumination light L1 emitted from the light source unit LS is transmitted through the ⁇ / 2 plate 21 so that the polarization direction is rotated in the y direction.
  • the ⁇ / 2 plate 21 is not necessary when the light source unit LS emits the illumination light L1 having the deflection direction in the y direction.
  • the expander lens 22 includes cylindrical lenses 22A and 22B facing each other. Since the cylindrical lenses 22A and 22B have no power in the y direction, the illumination light L1 has a constant width in the y direction. The width of the illumination light L1 in the z direction is enlarged or reduced according to the distance in the optical axis direction of the cylindrical lenses 22A and 22B. The expander lens 22 enlarges the width of the illumination light L1 in the z direction as an example by a factor of two.
  • the adjustment unit CL adjusts the incident illumination light L ⁇ b> 1 that has been expanded by the expander lens 22 in the width in the z direction.
  • the adjustment unit CL is disposed in the optical path between the light source unit LS and the objective lens 31.
  • the adjustment unit CL is disposed in the optical path between the ⁇ / 2 plate 21 or the expander lens 22 and the objective lens 31.
  • the adjustment unit CL may include a drive mechanism, and the light collection point may be adjusted by the movement of the adjustment unit CL.
  • the adjustment unit CL can be driven in the x direction, for example. In this case, even when a chip having a different position of the flow path 13 is used, it is possible to adjust so that the light collecting point is located in the flow path 13.
  • FIG. 9 is a partial detailed view of the adjustment unit CL and the fluid device C according to the embodiment.
  • the adjustment unit CL is configured by a cylindrical lens.
  • the adjustment portion CL has a minimum width in the z-direction of the illumination light L1 inside the flow path 13, and the passage region of the illumination light L1 at the position of the side face 16a on the irradiation light incident side of the flow path 13 is within the side face 16a.
  • the convergence angle is adjusted so as to be limited.
  • the adjustment portion CL has a minimum width in the z direction of the illumination light L1 inside the flow path 13, and the irradiation area of the illumination light L1 at the position of the side face 16a on the irradiation light incident side of the flow path 13 is within the side face 16a.
  • the illumination light L1 is adjusted to a convergence angle that condenses light.
  • the adjustment part CL is adjusted to the convergence angle which converges so that the passage area
  • the adjusting unit CL adjusts the illumination light L1 to a convergence angle such that the irradiation region of the illumination light L1 (irradiation light beam) at the position of the side surface 16b on the irradiation light emission side of the flow path 13 is condensed in the side surface 16b. . Further, the adjustment unit CL adjusts the convergence angle so that the irradiation region of the illumination light L1 at the position of the end surface 17 of the reservoir member 10 converges in the end surface 17. Further, the adjustment unit CL adjusts the convergence angle so that the illumination light L1 has a convergence point in the detection region in the flow path 13.
  • the illumination light beam of the illumination light L1 outside the focal depth of the detection unit 30 has a convergence angle that is smaller than the illumination light beam within the focal depth.
  • the above-described orthogonal surface includes the end surface 17 of the reservoir member 10, the side surface 16a on the irradiation light incident side of the flow path 13, or the side surface 16b on the irradiation light emission side of the flow path 13.
  • the convergence angle in the medium is ⁇
  • the wavelength of the illumination light L1 is ⁇
  • the beam width in the z direction at the position x and the convergence angle ⁇ is ⁇ (x, ⁇ )
  • the beam profile factor of the illumination light L1 is M2
  • the minimum width ⁇ 0 Assuming that the distance from the position in the x direction to the side surface 16a is xL, the following formula (3) and formula (4) must satisfy formula (5).
  • the beam width ⁇ (x, ⁇ ) included in the above equations (3) to (5) is 1 / e2 with respect to the peak value of the intensity of the illumination light L1. It is specified by the width. Even when the convergence angle ⁇ satisfies the expressions (1) to (3), the illumination light L1 having an intensity that is 1 / e2 or less with respect to the peak value is outside the beam width ⁇ (xL, ⁇ ). Therefore, when the convergence angle ⁇ is set, the beam width of the illumination light L1 having an intensity that is 1 / e2 or less with respect to the peak value is also taken into consideration.
  • FIG. 10 is a diagram schematically showing an optical path through which the illumination light L1 passes through the end face 17 of the reservoir member 10 and the side face 16a of the flow path 13.
  • the angle ⁇ 3 is the elevation angle of the illumination optical axis viewed from the focal plane F, and the counterclockwise direction from the focal plane F is the positive direction.
  • the incident angle and the emission angle at the interface, the inclination angle of the end surface 17 of the reservoir member 10 and the side surface 16a of the flow path 13 with respect to the yz plane, the illumination light flux in the material of the air / flow path device C and in the flow path The following relationship is established between the elevation angle with respect to the focal plane F, the medium outside the channel device C, the material of the channel device C, and the refractive index of the medium in the channel 13.
  • Incident angle / outgoing angle angle from the perpendicular to the end face 17 and the wall surface 16a Tilt angle: angle
  • the elevation angle ⁇ 3 of the illumination light L1 in the flow path 13 is expressed by the following equation (7).
  • the inclination angle of the end face 17 of the reservoir member 10 and the wall surface 16a of the flow path 13 and the elevation angle ⁇ 3 of the illumination light L1 are the refractive index n1 of the free space medium, the refractive index n2 of the material of the reservoir member 10 and the flow path 13
  • the refractive index n3 of the medium it is necessary to be selected, manufactured and adjusted so as to satisfy Expression (8).
  • the stage unit ST moves in the x direction, the y direction, and the z direction by driving the stage driving unit 60 shown in FIG.
  • the driving of the stage driving unit 60 is controlled by the control device 5.
  • the stage unit ST includes an installation surface STa on which the fluid device C is installed.
  • the installation surface STa is a surface parallel to the xy plane.
  • the installation surfaces STa are arranged at intervals in the y direction.
  • the installation surface STa supports both ends in the y direction where the lane 2 of the flow channel device C is not provided from the ⁇ Z side.
  • the region where the lane 2 is arranged is supported on the installation surface STa without hindering the observation from the ⁇ Z side by the detection unit 30.
  • stage part ST does not exist in the optical path of the illumination light L1 until the lane 2 in the fluid device C is irradiated, a part of the illumination light L1 incident on the fluid device C enters the stage part ST, which will be described later. Adversely affecting the particle detection.
  • the fixing pin 51 protrudes from the installation surface STa.
  • the fixing pin 51 includes two fixing pins 51 a that contact the long side of the fluid device C and one fixing pin 51 b that contacts the short side of the fluid device C.
  • the fixing pins 51a are arranged in the vicinity of both sides of the fluid device C in the y direction.
  • the fixing pin 51b contacts the short side located on the + y side.
  • a pressing piece 52 is provided at a corner located opposite to the corner where the fixing pin 51a and the fixing pin 51b located on the + y side are arranged.
  • the pressing piece 52 presses the fluid device C diagonally against the stage part ST.
  • the pressed fluid device C is fixed in a state where the fluid device C is positioned on the stage portion ST in the xy direction so that the flow path 13 (lane 2) is parallel to the y direction by contacting the fixing pins 51a and 51b.
  • the detection unit 30 includes an objective lens 31 and an imaging unit 32.
  • the objective lens 31 is disposed on the ⁇ Z side of the stage unit ST and the fluid device C. As shown in FIG. 9, the objective lens 31 is disposed at a position where the detection axis 31a passes through the center of the flow path 13 in the x direction.
  • the detection axis 31a is orthogonal to the optical axis of the illumination light L1.
  • the imaging unit 32 includes an EMCD (Electron Multiplying Charge Coupled Device) camera as an example, and captures an image of incident light.
  • the imaging unit 32 acquires image information of side scattered light that enters through the objective lens 31.
  • the transmission unit 40 transmits the image information captured by the imaging unit 32 to the control device 5.
  • the operation of the particle detection apparatus 100 includes an installation process, an introduction process, an irradiation process, and a detection process.
  • the installation process is a process of installing the fluid device C on the installation surface STa of the stage part ST. Specifically, as shown in FIG. 5, by pressing the fluid device C diagonally with the pressing piece 52, the fluid device C is pressed against the fixing pins 51a and 51b, and the flow path 13 (lane 2). Is placed on the installation surface STa in a state of being positioned on the stage portion ST so as to be parallel to the y direction.
  • the introducing step is a step of introducing a sample containing particles into the holding spaces 14A and 14B and the flow path 13 of the fluid device C.
  • a sample containing particles such as a sample containing particles
  • an exosome suspension in which exosomes are suspended in a buffer solution (medium) such as a phosphate buffer can be used as the sample.
  • the control device 5 drives the stage drive unit 60 so that the lane 2 to be detected is positioned on the optical path of the illumination light L 1 and the detection axis 31 a of the detection unit 30.
  • the control device 5 controls the power supply unit BT to apply an electric field to the electrodes 18A and 18B, and applies a force for causing the exosome to electrophores along the flow path 13. To do.
  • the control device 5 applies a voltage having an electric field strength of about 50 V / cm for about 10 seconds.
  • the moving direction of the exosome is parallel to the y direction.
  • the irradiation process is a process of irradiating the flow path 13 of the flow path device C with the illumination light L1 parallel to the x direction.
  • the irradiation unit 20 and the adjustment unit CL that irradiate the illumination light L1 have a constant width in the y direction, and have a sheet beam shape that converges in the z direction at a convergence angle ⁇ that satisfies the above-described equations (3) to (8).
  • Irradiation light L1 is irradiated.
  • the minimum beam thickness (beam width in the z direction) of the illumination light L1 is 10 ⁇ m.
  • the minimum beam thickness (beam width in the z direction) direction of the illumination light L1 is the z direction in FIGS.
  • the direction of the minimum beam thickness (beam width in the z direction) of the illumination light L1 is different from the optical axis direction and the flow path direction of the illumination light L1 on the incident surface (end surface 17 and side surface 16a). It is a direction orthogonal to the flow path direction.
  • the channel direction is a direction in which the channel 13 extends.
  • the flow path direction is a direction in which fluid flows through the flow path 13.
  • the irradiated illumination light L1 is one end surface (illumination light incident side end surface) 17 of the fluid device C, the side surface (illumination light incident side side surface) 16a of the flow channel 13, the inside of the flow channel 13, the side surface of the flow channel 13 ( The light passes through the illumination light emission side surface 16b and the other end surface (illumination light emission side end surface) 27 (see FIG. 5) of the fluid device C sequentially.
  • the illumination light L1 is irradiated in a direction orthogonal to the moving direction of the exosome. As shown in FIG.
  • the irradiated illumination light L1 converges so that the width in the z direction is minimized inside the flow path 13, and the irradiation light flux passage region at the position of the side surface 16 a of the flow path 13. Converges to be confined within the side surface 16a. Furthermore, the irradiated illumination light L1 converges so that the passage region of the irradiated light beam at the position of the side surface 16b on the illumination light exit side of the flow path 13 is limited to the side surface 16a.
  • the illumination light L1 is adjusted to a convergence angle such that the irradiation region at the position of the side surface 16a is condensed in the side surface 16a and the irradiation region at the position of the side surface 16b is condensed in the side surface 16b. Further, the irradiated illumination light L1 has a convergence point in the detection region of the detection unit 30 in the flow path 13.
  • the detection unit 30 observes (images) and detects the scattered light generated from the particles in the flow path 13 by irradiation of the illumination light L1 in parallel with the x direction. Since the detection axis 31a of the objective lens 31 in the detection unit 30 is orthogonal to the optical axis of the illumination light L1, the detection unit 30 detects side scattered light generated from the particles. The detection unit 30 detects light scattered toward the z direction perpendicular to the x direction by irradiation of the illumination light L1 irradiated in parallel with the x direction. The image of the particles in which the scattered light is observed is picked up by the image pickup unit 32. The transmission unit 40 transmits the image information captured by the imaging unit 32 to the control device 5.
  • the control device 5 comprehensively controls the particle detection system 1.
  • the control device 5 controls the movement of the stage unit ST and the fluid device C via the stage driving unit 60.
  • the control device 5 controls the power supply unit (application unit) BT to apply an electric field in the direction along the flow path 13 to the electrodes 18A and 18B.
  • the control apparatus 5 performs various determinations by processing the image captured by the particle detection apparatus 100. Details of the configuration of the control device 5 will be described with reference to FIGS. 11 to 16.
  • FIG. 11 is a diagram showing a schematic configuration of the control device 5 of the present embodiment.
  • the control device 5 includes a calculation unit 500 and a storage unit 520.
  • the storage unit 520 includes storage devices such as a flash memory, an HDD (Hard Disk Drive), a RAM (Random Access Memory), a ROM (Read Only Memory), and a register.
  • the storage unit 520 stores a program (firmware) executed by the calculation unit 500 in advance.
  • the storage unit 520 stores a calculation result obtained by performing the calculation process by the calculation unit 500.
  • the calculation unit 500 includes a CPU (Central Processing Unit) and performs various calculations.
  • the calculation unit 500 includes, as its functional units, an acquisition unit 501, an identification unit 502, a zeta potential determination unit 503, a particle diameter determination unit 504, a correlation unit 505, a state determination unit 506, and an evaluation unit 507. It has.
  • the acquisition unit 501 acquires an image captured by the particle detection device 100. Specifically, as described above, the imaging unit 32 of the particle detection apparatus 100 captures an image of side scattered light that is incident through the objective lens 31 and transmits image information of the captured image to the transmission unit 40. Output to. The acquisition unit 501 acquires the image information of the side scattered light image captured by the imaging unit 32 via the transmission unit 40. The acquisition unit 501 outputs the acquired image to the identification unit 502.
  • the identification unit 502 extracts a fine particle image from the image captured by the particle detection device 100. For example, the identification unit 502 extracts a fine particle image by performing known filter processing and pattern matching processing on the image supplied from the acquisition unit 501. At this time, the identification unit 502 may assign a particle number for each fine particle to the extracted fine particle image. When the identification target microparticle is an extracellular vesicle, the particle number may be an extracellular vesicle identifier. That is, the identification unit 502 may label the fine particles. This facilitates the correlation between the zeta potential ⁇ of the fine particles and the particle diameter d of the fine particles in the correlation section described later.
  • the identification unit 502 performs tracking on the labeled fine particle based on the difference between frames of the image captured by the particle detection device 100.
  • tracking refers to tracking changes with time in the coordinates of particles in an image. An example of the result of the identification unit 502 tracking fine particles is shown in FIG.
  • FIG. 12 is a diagram illustrating an example of the particle list LS1 stored in the storage unit 520.
  • the particle list LS1 stores the coordinates (X, Y) of the image of each fine particle at each time, with the row direction as the labeled particle number and the column direction as the imaging time.
  • the coordinates of each fine particle from the fine particle P1 to the fine particle Pn at each time from the time t0 to the time t50 are stored in the particle list LS1.
  • the zeta potential determination unit 503 determines the zeta potential ⁇ for each fine particle based on the result tracked by the identification unit 502. For example, the zeta potential determination unit 503 determines the zeta potential ⁇ 1 of the fine particle P1 based on the moving speed v1 of the fine particle P1 from the time t0 to the time t1 in the tracking result of the fine particle P1 performed by the identification unit 502. To do. The zeta potential determination unit 503 determines the zeta potential ⁇ based on the above equation (1). In this example, the dielectric constant ⁇ of the sample solution and the viscosity coefficient ⁇ of the sample solution are stored in the storage unit 520 in advance.
  • the zeta potential determination unit 503 is configured based on the permittivity ⁇ of the sample solution and the viscosity coefficient ⁇ of the sample solution stored in the storage unit 520 and the moving speed of the particles obtained from the tracking result by the identification unit 502. The zeta potential ⁇ is determined.
  • the particle diameter determination unit 504 determines the diameter of the fine particles based on the amount of movement of the fine particles due to Brownian motion in the sample solution and the above equation (2).
  • the particle size determination unit 504 determines the particle size of the fine particles P1.
  • the Boltzmann constant k and the absolute temperature T of the sample solution are stored in the storage unit 520 in advance.
  • the particle diameter determination unit 504 calculates the movement amount of the fine particles P1 based on the result tracked by the identification unit 502.
  • the particle size determination unit 504 determines the particles of the fine particles P1 based on the calculated movement amount of the fine particles P1, the Boltzmann constant k and the absolute temperature T stored in the storage unit 520, and the above equation (2).
  • the diameter d1 is determined.
  • the correlation unit 505 associates the zeta potential ⁇ of the fine particles determined by the zeta potential determination unit 503 with the particle size d of the fine particles determined by the particle size determination unit 504. Specifically, the first zeta potential ⁇ 1 determined for the first fine particles in the zeta potential determination unit 503, and the first particle diameter d1 determined for the first fine particles in the particle size determination unit 504 Are correlated with each other as data on the first fine particles in the correlation unit 505.
  • FIG. 13 shows an example of the particle correlation list LS2 that is a result associated with the correlation unit 505.
  • FIG. 13 is a diagram illustrating an example of the particle correlation list LS2 stored in the storage unit 520.
  • the particle diameter d and the zeta potential ⁇ are associated with each particle number assigned by the identification unit 502.
  • the correlation unit 505 associates the particle diameter d1 of the fine particle P1 with the zeta potential ⁇ 1 of the fine particle P1 and stores it as particle correlation information PC1 (d1, ⁇ 1) in the particle correlation list LS2 for the fine particle P1. Further, the correlation unit 505 associates the particle diameter d2 of the fine particle P2 with the zeta potential ⁇ 2 of the fine particle P2 and stores the particle P2 in the particle correlation list LS2 as particle correlation information PC2 (d2, ⁇ 2). In this way, the correlation of the state of the fine particles existing in the medium can be determined.
  • the state determination unit 506 determines the state of the fine particles based on the particle correlation list LS2 generated by the correlation unit 505.
  • the storage unit 520 stores reference range information indicating the reference range of the particle diameter d and the reference range of the zeta potential ⁇ .
  • state determination by the state determination unit 506 a case will be described in which it is determined whether or not the microparticles identified by the identification unit 502 are exosomes in a sample containing particles other than exosomes.
  • the characteristics of exosomes are microparticles with a particle size of about 30 to 200 nm, and the presence of chaperone molecules Hsc70, Hsc90 and tetraspanins (CD9, CD63, CD81) as constituent factors.
  • the storage unit 520 stores a threshold value Thd of particle diameter as reference range information.
  • the storage unit 520 stores a threshold value Th ⁇ of zeta potential as reference range information.
  • the storage unit 520 may be rephrased as a reference storage unit. An example of the threshold value Thd and the threshold value Th ⁇ is shown in FIG.
  • FIG. 14 is a diagram illustrating an example of threshold values stored in the storage unit 520 of the present embodiment.
  • the particle diameter of exosome is about 30 to 100 nm in diameter, and among the particles to be determined, the particle diameter of particles other than exosome exceeds 100 nm in diameter.
  • the zeta potential ⁇ of the exosome is equal to or lower than the threshold Th ⁇ and the zeta potential ⁇ of fine particles other than the exosome exceeds the threshold Th ⁇ .
  • the state determination unit 506 can determine the particle based on the particle diameter of the particle and the zeta potential ⁇ of the particle.
  • the determination of fine particles performed by the state determination unit 506 may be paraphrased as identification of fine particles.
  • the storage unit 520 stores 100 nm as the particle size threshold Thd.
  • the storage unit 520 stores ⁇ 6 mV as the threshold value Th ⁇ of the zeta potential.
  • the state determination unit 506 is a particle whose particle diameter d is equal to or less than the threshold Thd in the particle correlation information PC stored in the particle correlation list LS2, and the zeta potential ⁇ is equal to or less than the threshold Th ⁇ .
  • a microparticle is determined to be an exosome.
  • the state determination unit 506 among the particle correlation information PC stored in the particle correlation list LS2, fine particles having a particle diameter d exceeding the threshold value Thd, or fine particles having a zeta potential ⁇ exceeding the threshold value Th ⁇ . Is determined not to be an exosome.
  • the particle diameter of exosome is about 30 to 100 nm, and among the fine particles to be determined, the particle diameter of fine particles other than exosome may exceed 200 nm.
  • the state determination unit 506 can determine the state of the fine particles based only on the particle diameter.
  • fine particles other than exosomes may be included in the diameter range of 100 to 200 nm.
  • the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether or not the fine particle is an exosome.
  • a single exosome may not be contained in the range whose diameter is larger than 200 nm.
  • the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether or not the microparticle is a single exosome.
  • a range in which the diameter is larger than 200 nm may include fine particles in which a plurality of single exosomes are aggregated.
  • the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether the microparticle is a single exosome or an aggregated exosome.
  • the threshold value serving as the reference value stored in the reference storage unit can be used as a factor for determining the state of the fine particles.
  • the state determination unit 506 determines whether or not the exosome has reacted with the antibody when the microparticles identified by the identification unit 502 are exosomes.
  • the storage unit 520 stores a threshold value Th ⁇ of zeta potential as reference range information. As described above, the zeta potential of the antibody-exosome complex is positively shifted compared to the zeta potential of exosome alone. In this case, the storage unit 520 stores a zeta potential (eg, ⁇ 6 mv) between the zeta potential of the exosome alone and the zeta potential of the antibody-exosome complex as the zeta potential threshold Th ⁇ . .
  • the state determination unit 506 determines that, among the particle correlation information PC stored in the particle correlation list LS2, the fine particles whose zeta potential of the fine particles is less than the threshold Th ⁇ is a single exosome that has not reacted with the antibody. judge. On the other hand, in the particle correlation information PC stored in the particle correlation list LS2, the state determination unit 506 determines that the microparticles whose microparticle zeta potential is greater than or equal to the threshold Th ⁇ are antibody-exosome complexes. .
  • the threshold value Th ⁇ for example, ⁇ 6 mV
  • the threshold value Th ⁇ ( ⁇ 6 mV) of the zeta potential can be used as one element for determining whether the microparticle is a single exosome.
  • the state determination unit 506 can also determine the state of the fine particles by combining the threshold value of the particle diameter d and the threshold value of the zeta potential ⁇ .
  • the antibody-exosome complex has a low zeta potential compared to a single exosome. For this reason, the Coulomb force acting between the microparticles is weaker in the antibody-exosome complex than in the single exosome.
  • the Coulomb force acting between the fine particles acts as a repulsive force that keeps the fine particles apart. That is, the antibody-exosome complex has less repulsive force acting between the microparticles than the single exosome. For this reason, the antibody-exosome complex tends to aggregate more easily than a single exosome.
  • the particle diameter determination unit 504 shifts the particle diameter d to be larger than that in the case where the particles are not aggregated by determining a plurality of aggregated fine particles as one fine particle.
  • a case where a fine particle having a particle diameter d of 200 nm or less is determined to be an exosome will be described as an example.
  • the particle size determination unit 504 may determine that the antibody-exosome complex is a fine particle having a diameter exceeding 200 nm.
  • the state determination unit 506 determines only by the particle diameter d, the particle diameter d of the antibody-exosome complex exceeds the threshold value Thd of the particle diameter d whether or not the antibody is an exosome. -It may be determined that the exosome complex is not an exosome. Therefore, the state determination unit 506 determines that a fine particle having a particle diameter d of 200 nm or less is an exosome, and the zeta potential ⁇ is equal to or less than a threshold Th ⁇ even if the particle diameter d is greater than 200 nm. Is determined to be an exosome. That is, the state determination unit 506 determines whether or not the fine particle is an exosome by combining the threshold value Thd of the particle diameter d and the threshold value Th ⁇ of the zeta potential ⁇ .
  • an antibody that specifically binds to the exosome such as tetraspanin (CD9, CD81, etc.) can be used. That is, it is possible to determine whether or not the microparticle is an exosome based on the respective changes in the zeta potential ⁇ and the particle diameter d caused by causing the antibody to act on the exosome.
  • the particle detection system 1 has an advantage that fine particles can be evaluated by variously combining the above-described evaluation conditions based on the zeta potential ⁇ and the particle diameter d.
  • the state determination unit 506 can determine the state of the fine particles based on the result of tracking by the identification unit 502 after combining the threshold value Thd of the particle diameter d and the threshold value Th ⁇ of the zeta potential ⁇ . . Specifically, the state determination unit 506 tracks the progress until the antibody reacts with a single exosome and further aggregates the antibody-exosome complexes based on the result of tracking by the identification unit 502. Specifically, the state determination unit 506 moves the particle diameter d and the zeta potential ⁇ of each fine particle from any of the regions DM1 to DM4 shown in FIG.
  • the state of the fine particles is determined depending on whether or not As an example, the state determination unit 506 determines that when a microparticle (for example, a single exosome) present in the region DM3 moves to the region DM2, the exosome reacts with the antibody and changes to an antibody-exosome complex. judge. Further, when the exosome moves from the region DM2 to the region DM1, the state determination unit 506 determines that the antibody-exosome complexes are aggregated.
  • a microparticle for example, a single exosome
  • Evaluation unit 507 evaluates the quality of the fine particles.
  • the evaluation unit 507 ranks the state of the fine particles into A rank, B rank, and C rank based on the state of the fine particles determined by the state determination unit 506.
  • the A rank is a case where both the particle diameter d and the zeta potential ⁇ of the fine particles are included in the reference range.
  • the rank B is a case where either one of the particle diameter d and the zeta potential ⁇ of the fine particles is not included in the reference range.
  • the C rank is a case where neither the particle diameter d of the fine particles nor the zeta potential ⁇ is included in the reference range.
  • the evaluation unit 507 evaluates whether or not the microparticle is a single exosome.
  • the evaluation unit 507 determines that the rank of the fine particles is rank A when the fine particles are present in the region DM3. Further, the evaluation unit 507 determines that the rank of the fine particles is rank B when the fine particles are present in the region DM2 or the region DM4. The evaluation unit 507 determines that the rank of the fine particles is rank C when the fine particles are present in the region DM1.
  • FIG. 15 is a diagram illustrating an example of the operation of the control device 5.
  • the particle detection apparatus 100 captures an image of side scattered light at a predetermined time interval.
  • the acquisition unit 501 acquires images captured by the imaging unit 32 of the particle detection device 100 one by one from the particle detection device 100 (step S10).
  • This image includes an image of fine particles that are electrophoresed in the migration channel 150.
  • the fine particle image includes an exosome image.
  • the identification unit 502 extracts a fine particle image from the image acquired in step S10, and assigns a unique particle number to each fine particle. That is, the identification unit 502 labels the fine particles (step S20).
  • the identification unit 502 determines whether or not labeling has been completed for all captured images (step S30). If it is determined that the labeling has not been completed for all captured images (step S30; NO), the identification unit 502 returns the process to step S10 and performs the labeling for the next image. If it is determined that the labeling has been completed for all captured images (step S30; YES), the identification unit 502 advances the process to step S40 and performs tracking for the identified fine particles.
  • the zeta potential determination unit 503 determines the zeta potential ⁇ for each fine particle based on the result tracked by the identification unit 502 (step S50). Further, the particle size determination unit 504 determines the particle size for each fine particle based on the result of tracking by the identification unit 502 (step S60). Note that the order of step S50 and step S60 may be reversed or may be executed in parallel.
  • correlation section 505 associates the zeta potential ⁇ of the fine particles determined by zeta potential determination section 503 with the particle diameter d of the fine particles determined by particle diameter determination section 504 (step SS70).
  • the correlation unit 505 generates a particle correlation list LS2 indicating the associated result, and stores the generated particle correlation list LS2 in the storage unit 520.
  • the correlation unit 505 determines that the association has not been completed for all the fine particles (step S80; NO)
  • the correlation unit 505 returns the process to step S40.
  • the correlation unit 505 determines that the association has been completed for all the fine particles (step S80; YES)
  • the correlation unit 505 advances the process to step S90.
  • the state determination unit 506 and the evaluation unit 507 perform particle state determination and evaluation based on the particle correlation list LS2 generated in step S70.
  • the particle detection apparatus 100 is an example of an apparatus for measuring the zeta potential of extracellular vesicles, and can be a device capable of measuring the zeta potential of individual extracellular vesicles in a population of extracellular vesicles. Can be used without any particular restrictions.
  • the particle detector configured as described above, or other zeta potential measuring device the zeta potential of individual extracellular vesicles in a population of extracellular vesicles is measured, and the standard deviation of those zeta potentials is calculated. It can be calculated as the standard deviation of the zeta potential of the population of extracellular vesicles.
  • the standard deviation of the zeta potential of the extracellular vesicle calculated as described above is 5 mV or less, it is determined that the group is the extracellular vesicle group of the present embodiment.
  • the population of extracellular vesicles of this embodiment is a population of extracellular vesicles having a standard deviation of zeta potential of 5 mV or less and high homogeneity with respect to the state of extracellular vesicles, particularly the surface state of extracellular vesicles. is there. Therefore, it can be used for various uses such as pharmaceuticals, cosmetics, and foods.
  • the extracellular vesicle population of this embodiment can be suitably used as a pharmaceutical product that requires particularly high homogeneity because the quality of the extracellular vesicles constituting the population is uniform.
  • Examples of the pharmaceutical use include, but are not limited to, use as a carrier encapsulating a drug and use of a specific cell-derived exosome such as a mesenchymal stem cell as a drug.
  • a specific cell-derived exosome such as a mesenchymal stem cell as a drug.
  • the zeta potential is measured in a state in which a specific binding substance is bound to an extracellular vesicle, the extracellular smallness with high homogeneity particularly with respect to the expression state of the molecule to which the specific binding substance is bound.
  • a population of cells can be obtained.
  • the membrane surface of an extracellular vesicle is modified with a molecule that targets a disease site or the like (for example, an antibody against a cancer cell membrane surface antigen)
  • the zeta potential can be reduced using a specific binding substance for the molecule.
  • Measurement may be performed to obtain a population of extracellular vesicles having a standard deviation of the zeta potential of 5 mV or less.
  • Extracellular vesicles constituting such a population of extracellular vesicles are particularly homogeneous with respect to the state of modification of the membrane surface by the molecule. Therefore, it can be suitably used as a carrier for DDS to the disease site.
  • the present invention provides a method for producing a population of extracellular vesicles.
  • the method of the present embodiment includes (a) a step of synchronizing the cell cycle of a plurality of cells, and (b) a medium of the plurality of cells substantially not including extracellular vesicles after the step (a). Replacing the medium; (c) culturing the plurality of cells in the medium replaced; and (d) recovering a population of extracellular vesicles from the medium after the step (c). And including.
  • Step (a) is a step of synchronizing the cell cycle of a plurality of cells.
  • the plurality of cells are preferably the same type of cells. By culturing the same type of cells, extracellular vesicles of uniform quality can be obtained.
  • the cell type is not particularly limited as long as it releases extracellular vesicles.
  • Examples of cells include various disease cells such as tumor cells; immune cells such as dendritic cells, T cells and B cells; various tissue cells such as nerve cells; fat cells; mesenchymal stem cells and hematopoietic stem cells. Examples include, but are not limited to, stem cells; pluripotent stem cells such as ES cells and iPS cells; germ cells and the like.
  • the biological species from which the cells are derived is not particularly limited.
  • the biological species from which the cells are derived is not particularly limited, and examples thereof include cells of humans and mammals other than humans (for example, mice, guinea pigs, monkeys, dogs, cats, cows, horses, pigs, etc.).
  • the cells may be appropriately selected according to the use of the exosome to be produced.
  • human mesenchymal stem cells and the like can be selected.
  • “More cells” if two or more cells is not particularly limited, for example, 10 2 or more, 10 3 or more, may be a cell, such as a 10 4 or more.
  • the plurality of cells may be, for example, 10 2 to 10 15 cells, 10 3 to 10 12 cells, or 10 4 to 10 10 cells.
  • the cell cycle is divided into an interphase and an M phase, and the interphase is further divided into a G1 phase, an S phase, and a G2 phase (see FIG. 16).
  • the cell cycle for synchronizing a plurality of cells is not particularly limited, and may be any of G1 phase, S phase, G2 phase, and M phase. Further, it may be synchronized with the boundary of two periods such as the boundary of G1 period / S period.
  • the method for synchronizing the cell cycle of the plurality of cells is not particularly limited, and a known method can be used. Examples of such a method include a method of culturing in a medium containing a cell cycle synchronizer, a method of culturing cells in a confluent state, a method of culturing cells in a serum-starved state, and a thymidine block method.
  • the “cell cycle synchronizer” is an agent having an action of synchronizing the cell cycle of a plurality of cells.
  • the cell cycle synchronizer include a drug having an action of stopping the progression of the cell cycle in a specific cell cycle.
  • a well-known thing can be especially used for a cell cycle synchronizing agent without a restriction
  • leptomycin A, leptomycin B, etc. are mentioned as a cell cycle synchronizer which synchronizes a cell to G1 phase.
  • a cell cycle synchronizer is added to a culture medium, and a plurality of cells are cultured. What is necessary is just to select a culture medium suitably according to the kind of cell.
  • a known human cell culture medium can be used without any particular limitation.
  • An example of the human cell culture medium is RPMI medium to which fetal bovine serum (FBS) or the like is added.
  • the culture time in a medium containing a cell cycle synchronizer may be appropriately selected according to the cell type. It is preferable to culture for a period of time longer than the cell cycle proceeds for one cycle. Examples of the culture time include 10 hours or more, 15 hours or more, or 20 hours or more.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 5 cycles, 4 cycles, 3 cycles, or 2 cycles, etc. Is mentioned. Specific examples of the upper limit of the culture time include, for example, within 50 hours, within 40 hours, or within 30 hours. As an example, the culture time can be 24 hours.
  • the cell concentration at the start of the culture is not particularly limited, and may be, for example, 10 3 to 10 10 cells / mL, 10 3 to 10 9 cells / mL, 10 4 to 10 9 cells / mL, and the like.
  • the cell culturing conditions are not particularly limited, and conditions generally used for culturing the cells may be used according to the cell type.
  • the temperature conditions include 25 to 40 ° C., 30 to 37 ° C. and the like.
  • the “confluent state” refers to a state in which cells reach a concentration capable of growing in a culture container and cell growth is almost stopped. By culturing cells in a confluent state, the cell cycle can be synchronized with the G1 phase.
  • the cells when cells are cultured in a culture container such as a petri dish, the cells proliferate and reach a confluent state.
  • a confluent state For example, when culturing in a petri dish or the like, it can be determined that a confluent state has been reached when cells have spread throughout the petri dish. In this state, the cell cycle can be synchronized by continuing the culture.
  • the culture time after reaching the confluent state is preferably cultivated for at least the time required for one cycle of the cell cycle, as described above. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized.
  • the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. .
  • Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours.
  • the culture time can be 24 hours.
  • the culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium not containing cell cycle synchronizer is used.
  • the culture in the serum starvation state may be performed by a known method by culturing cells in a medium not containing serum.
  • a medium not containing serum can be prepared, for example, by adding no serum in the composition of the medium appropriately selected according to the cell type.
  • the cell cycle can be synchronized with the G1 phase.
  • the culture time in the serum starvation state is preferably cultivated for a time longer than the time required for one cycle of the cell cycle as described above. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. . Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours. As an example, the culture time can be 24 hours.
  • the culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium not containing serum is used.
  • the thymidine block may be performed by a known method by culturing cells in a medium containing an excessive amount of thymidine. By performing thymidine blocking, the cell cycle can be synchronized to the S phase. Examples of the concentration of thymidine in the medium include 1 to 5 mM, 1.5 to 4 mM, and 2 to 3 mM. The medium may be appropriately selected according to the cell type, except that thymidine is added in excess. As in the above, the culture time in the medium containing excess thymidine is preferably cultivated for at least the time required for one cycle of the cell cycle. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. . Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours. As an example, the culture time can be 24 hours.
  • the cell cycle may be synchronized using the double thymidine block method.
  • the double thymidine block method can also be performed by a known method.
  • thymidine block method thymidine block is performed twice. For example, after culturing in a medium containing excess thymidine for a certain period (for example, about 10 to 30 hours), the medium is changed, and culturing is performed for a certain period (for example, about 6 to 20 hours) in a medium not containing thymidine. Thereafter, the cell cycle can be synchronized with the boundary between G1 phase / S phase by culturing again in a medium containing excess thymidine.
  • the culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium containing excess thymidine is used.
  • Step (b) is a step of replacing the medium of the plurality of cells with a medium substantially free of extracellular vesicles after the step (a).
  • the medium can be exchanged, for example, by removing the culture supernatant from the culture vessel in step (a) and adding a medium substantially free of extracellular vesicles. Further, after removing the medium supernatant, the cells may be washed about 1 to 3 times with a medium substantially free of extracellular vesicles. When the cells are floating cells or the like, the cells may be collected by centrifugation, filter filtration, etc., washed appropriately, and seeded in a medium substantially free of extracellular vesicles. .
  • the “medium substantially free of extracellular vesicles” is a medium substantially free of extracellular vesicles in the medium components. “Substantially free of extracellular vesicles” means that the content is negligible even if extracellular vesicles are contained, or no extracellular vesicles.
  • the concentration of extracellular vesicles contained in the medium is, for example, about 0 to 10 cells / mL, preferably about 0 to 5 cells / mL, more preferably Examples of the medium include about 0 to 3 / mL, more preferably about 0 to 1 / mL, and particularly preferably about 0 to 0.5 / mL.
  • the culture medium contains both extracellular vesicles released from the cells and extracellular vesicles originally contained in the medium. It is not possible to obtain extracellular vesicles with uniform size. Therefore, in this step, the cell culture medium is replaced with a medium that is substantially free of extracellular vesicles. By this step, it is possible to remove extracellular vesicles released before the cell cycle is synchronized, and to eliminate the introduction of extracellular vesicles from the medium.
  • the method for producing a medium substantially free of extracellular vesicles is not particularly limited, and known methods for recovering extracellular vesicles can be applied.
  • extracellular vesicles in the medium can be removed by performing ultracentrifugation, ultrafiltration, or the like. Therefore, it can also be said that the medium substantially free of extracellular vesicles is a medium that has been subjected to a treatment for removing extracellular vesicles in the medium after the medium has been adjusted.
  • Step (c) is a step of culturing the plurality of cells in the medium exchanged.
  • the cells can be cultured in the same manner as described in the above step (a) except that the cells are cultured in a medium not containing extracellular vesicles.
  • the culture time in this step may be a time sufficient for the cells to release extracellular vesicles. For example, it can be 30 minutes or longer, 1 hour or longer, 1.5 hours or longer. Further, for example, it may be 30 minutes to 5 hours, 1 to 4 hours, 1.5 to 3 hours, and the like. For example, the culture time can be 2 hours.
  • Step (d) is a step of collecting a population of extracellular vesicles from the medium after step (c).
  • Extracellular vesicles can be recovered, for example, from the culture supernatant after step (c).
  • the cells and the culture supernatant can be separated by centrifuging the culture solution or filtering the filter.
  • the method for recovering extracellular vesicles from the culture supernatant is not particularly limited, and a known method can be used.
  • methods for recovering extracellular vesicles include ultracentrifugation, ultrafiltration, continuous flow electrophoresis, chromatography and the like. In this way, a population of extracellular vesicles can be obtained.
  • the population of extracellular vesicles obtained by the production method of the present embodiment is a population of extracellular vesicles released from cells synchronized in cell cycle and of uniform quality.
  • the group of extracellular vesicles having uniform quality is, for example, a group of extracellular vesicles in which the standard deviation of the zeta potential of the extracellular vesicles is an arbitrary threshold value or less.
  • the extracellular vesicles whose standard deviation of zeta potential of extracellular vesicles is 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, further preferably 3.5 mV or less, particularly preferably 3 mV or less. It is a group of.
  • the population of extracellular vesicles obtained by the production method of the present embodiment has the same quality as described above, it is suitable for use as a drug for DDS carriers and regenerative medicine.
  • the manufacturing method of this embodiment may include an optional step in addition to the steps (a) to (d).
  • an arbitrary process is not specifically limited, For example, (e) The process of measuring the zeta potential of the extracellular vesicle contained in the collect
  • the production method of this embodiment may include a step of measuring the zeta potential of extracellular vesicles contained in the population of extracellular vesicles collected in step (d).
  • the zeta potential of the extracellular vesicle can be measured by a known method. Examples of the method for measuring the zeta potential of extracellular vesicles include the method described in the above “ ⁇ Extracellular vesicle population>”.
  • the manufacturing method of the present embodiment may further include a step of (f) calculating a standard deviation of the zeta potential obtained by the step (e). Further, (g) a step of selecting a population of extracellular vesicles in which the standard deviation is not more than a predetermined threshold value may be included.
  • the standard deviation threshold include 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, still more preferably 3.5 mV or less, and particularly preferably 3 mV or less.
  • a population of extracellular vesicles having uniform quality can be obtained by steps (a) to (d).
  • the quality can be further improved by carrying out steps (e) to (g).
  • a group of extracellular vesicles with uniform thickness can be obtained.
  • the present invention provides a method for assessing the quality of a population of extracellular vesicles.
  • the method of this embodiment includes (a) a step of measuring zeta potential of a plurality of extracellular vesicles contained in a population of extracellular vesicles, and (b) a standard of zeta potential measured in the step (a). And (c) evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b).
  • Step (a) is a step of measuring the zeta potential of a plurality of extracellular vesicles contained in the population of extracellular vesicles. Measurement of the zeta potential of individual extracellular vesicles in a population of extracellular vesicles can be performed by a known method. Examples of the method for measuring the zeta potential of extracellular vesicles include the method described in the above “ ⁇ Extracellular vesicle population>”.
  • Step (b) is a step of calculating the standard deviation of the zeta potential measured in step (a).
  • the standard deviation of the zeta potential can be calculated by the method described above in “ ⁇ population of extracellular vesicles>”.
  • Step (c) is a step of evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b). For example, when the standard deviation threshold is set based on the type of cell from which the extracellular vesicle is derived, the use of the extracellular vesicle, etc., and the standard deviation calculated in step (b) is equal to or less than the threshold It can be evaluated that the uniformity of the extracellular vesicle population is high. That is, it can be determined that the quality of the population of extracellular vesicles is high (the quality is uniform). Examples of the standard deviation threshold include 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, still more preferably 3.5 mV or less, and particularly preferably 3 mV or less.
  • the method of the present embodiment may further include a step of selecting a population of extracellular vesicles evaluated as having high uniformity by the step (c).
  • the method may include a step of discarding a population of extracellular vesicles evaluated as having low uniformity by the step (c).
  • the quality of the extracellular vesicle population can be managed, and the extracellular vesicle population with uniform quality can be maintained.
  • the present invention provides a composition comprising a plurality of extracellular vesicles, wherein the standard deviation of the zeta potential of the extracellular vesicles contained in the composition is 5 mV or less. To do.
  • the standard deviation of the zeta potential of the extracellular vesicles contained in the composition of this embodiment is 5 mV or less.
  • the standard deviation of the zeta potential is preferably 4.5 mV or less, more preferably 4 mV or less, preferably 3.5 mV or less, and more preferably 3 mV or less.
  • the plurality of extracellular vesicles may be two or more, and is not particularly limited.
  • the number of extracellular vesicles is 10 3 to 10 15 , 10 4 to 10 12 or more, 10 5 to 10 10 extracellular vesicles. May be.
  • the plurality of extracellular vesicles contained in the composition of the present embodiment is the population of extracellular vesicles of the above-described embodiment described in the above “ ⁇ population of extracellular vesicles>”.
  • composition of the present embodiment may contain an arbitrary component in addition to a plurality of extracellular vesicles.
  • arbitrary components are not specifically limited, For example, various buffer solutions (physiological saline, a phosphate buffer, a HEPES buffer etc.), a cell culture solution, etc. are mentioned.
  • composition of the present embodiment may be a pharmaceutical composition, cosmetics, food (including functional food, health food, etc.) and the like.
  • composition of this embodiment is a pharmaceutical composition, cosmetics, food, or the like, various components described later may be included depending on the application.
  • the present invention provides a pharmaceutical composition comprising the population of extracellular vesicles of the above embodiment.
  • the population of extracellular vesicles of the above embodiment, or the population of extracellular vesicles produced by the production method of the above embodiment (hereinafter sometimes collectively referred to as “the population of extracellular vesicles”). Due to its high uniformity, it can be contained in a pharmaceutical composition.
  • the population of extracellular vesicles of the above-described embodiment can be used, for example, for a carrier containing a drug (for example, a carrier of DDS) or regenerative medicine.
  • the pharmaceutical composition of this embodiment may contain other components in addition to the population of the present extracellular vesicles.
  • it may contain at least one pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the administration subject. By “not exhibiting substantial toxicity” is meant that the ingredient is not toxic to the administered subject at the doses normally used.
  • Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredients that are typically considered inactive ingredients.
  • the pharmaceutically acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (for example, antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. Can be mentioned.
  • the solvent include water, physiological saline, phosphate buffer, HEPES buffer, cell culture medium, DMSO, dimethylacetamide, ethanol, glycerol, mineral oil, and the like.
  • a pharmaceutically acceptable carrier may be used alone or in combination of two or more.
  • components commonly used in the pharmaceutical field can be used without particular limitation.
  • the pharmaceutical composition of the present embodiment includes, for example, a solubilizing agent, a suspending agent, an isotonic agent, a buffer, a pH adjusting agent, an excipient, a stabilizer, an antioxidant, an osmotic pressure adjusting agent, and an antiseptic.
  • Coloring agents, fragrances and the like may be included. These may be used alone or in combination of two or more.
  • the pharmaceutical composition of this embodiment may contain the chemical
  • medical agent is not specifically limited, What is necessary is just to select suitably according to the use of the pharmaceutical composition of this embodiment.
  • Examples of the drug include anticancer agents, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, stimulus relieving agents, analgesics, cell activators, plants and animals. ⁇ Microbial extract, antipruritics, anti-inflammatory analgesics, antifungals, antihistamines, hypnotic sedatives, tranquilizers, antihypertensives, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptics, coronary vasodilation Examples include, but are not limited to, agents, herbal medicines, antipruritic agents, and keratin softening release agents. A medicine may be used individually by 1 type and may use 2 or more types together. The drug may be encapsulated in, for example, extracellular vesicles that constitute the population of the present extracellular vesicles.
  • the dosage form of the pharmaceutical composition of the present embodiment is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation.
  • Examples of the dosage form of the pharmaceutical composition of the present embodiment include orally administered dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, and emulsions, or Examples include dosage forms administered parenterally such as injections, suppositories, and external preparations for skin.
  • the pharmaceutical composition of these dosage forms can be formulated according to a conventional method (for example, a method described in the Japanese Pharmacopoeia).
  • the administration route of the pharmaceutical composition of this embodiment is not particularly limited, and can be administered by oral or parenteral routes.
  • the parenteral route includes all routes other than oral administration such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, ophthalmic, intracerebral, intrarectal, intravaginal, intraperitoneal, etc. To do. Administration may be local or systemic.
  • the pharmaceutical composition of the present embodiment can be administered in a single dose or multiple doses, and the administration period and interval thereof include the type of drug, the type and condition of the disease, the administration route, the age of the administration subject, It can be appropriately selected depending on body weight and sex.
  • the administration interval can be, for example, 1 to 3 times a day, every 3 days, every week, etc.
  • the dosage of the pharmaceutical composition of the present embodiment can be appropriately selected depending on the type of drug, the type and condition of the disease, the administration route, the age, weight and sex of the administration subject.
  • the dosage of the pharmaceutical composition of the present embodiment can be a therapeutically effective amount of the drug contained in the pharmaceutical composition, for example, about 0.01 to 1000 mg per kg body weight at a time, 0.1 to 500 mg. About 0.1 to 100 mg.
  • the present invention provides a cosmetic product comprising the population of extracellular vesicles of the above embodiment.
  • the cosmetic product of this embodiment may contain other components as appropriate in addition to the population of the present extracellular vesicles.
  • the cosmetic of this embodiment can be manufactured according to a known method according to the type of cosmetic.
  • Other components are not particularly limited, and examples thereof include pharmaceutically acceptable carriers. Examples of the pharmaceutically acceptable carrier are the same as those exemplified in the above “ ⁇ Pharmaceutical composition>”.
  • the cosmetics of this embodiment may use materials known as cosmetic additives as other components. Other components may be used alone or in combination of two or more.
  • the cosmetic product of this embodiment may contain a drug (active ingredient) having a cosmetic effect or the like.
  • medical agent is not specifically limited, What is necessary is just to select suitably according to the use of the cosmetics of this embodiment.
  • the agent include whitening materials, ultraviolet absorbers, hair-growth agents, astringents, anti-wrinkle agents, anti-aging agents, tanning agents, antiperspirants, moisturizers, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, Examples include, but are not limited to, inflammatory agents, blood circulation promoters, stimulants, hormones, stimulant mitigators, cell activators, plant / animal / microbe extracts, herbal medicines, antipruritic agents, keratin softening release agents, and the like.
  • a medicine may be used individually by 1 type and may use 2 or more types together.
  • the drug may be encapsulated in, for example, extracellular vesicles that constitute the population of the present extracellular vesicles.
  • the type of cosmetic product is not particularly limited.
  • cosmetics include basic cosmetics such as lotions, emulsions, lotions, creams, gels, sunscreens, packs, masks, and cosmetics; makeup cosmetics such as foundations, makeup bases, lipsticks, lip glosses, and blushers Cleaning agents such as facial cleansers, body shampoos and cleansing agents; hair cosmetics such as shampoos, rinses, hair conditioners, treatments, hair styling agents; and body cosmetics such as body powders and body lotions, but are not limited to these Not.
  • the cosmetic product of the present embodiment can be used in the same manner as a normal cosmetic product, depending on the use of the cosmetic product.
  • the present invention provides a food product comprising the population of extracellular vesicles of the above embodiment.
  • the food of this embodiment may contain other components as appropriate in addition to the population of the present extracellular vesicles.
  • the food of this embodiment can be produced according to a known method according to the type of food.
  • Other components are not particularly limited, and for example, materials known as food additives may be used as other components.
  • Other components may be used alone or in combination of two or more.
  • the type of food is not particularly limited.
  • Examples of food include various types of noodles such as buckwheat, udon, harusame, Chinese noodles, instant noodles, cup noodles; carbohydrates such as bread, flour, rice flour, hot cakes, mashed potatoes; green juices, soft drinks, carbonated drinks, Beverages such as nutritional drinks, fruit drinks, vegetable drinks, lactic acid drinks, milk drinks, sports drinks, tea and coffee; bean products such as tofu, okara and natto; various soups such as curry roux, stew roux and instant soup; ice Frozen confectionery such as cream, ice sherbet, shaved ice; sweets such as candy, cookies, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, and other baked confectionery; kamaboko, hampen, ham, sausage Processed fishery and livestock products such as processed milk, fermented milk, butter, cheese, yogurt and other milk Products; salad oil, tempura oil, margar
  • the food of this embodiment may be health food, functional food, or the like. In this case, it may be formulated into a dry powder, granule, tablet, jelly, drink or the like by a known formulation method.
  • the food of this embodiment may be taken in the same way as normal food.
  • HL60 cells which are human acute myeloid leukemia cells, were cultured in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS).
  • RPMI Roswell Park Memorial Institute
  • FBS fetal bovine serum
  • EV removal medium in which EV in FBS was removed by ultracentrifugation was used.
  • EV purification from the collected culture supernatant was performed according to the following procedure. First, to remove large particles such as cell debris and microvesicles, centrifugation was performed at 300 ⁇ g for 10 minutes, 2,000 ⁇ g for 20 minutes, and 10,000 ⁇ g for 100 minutes.
  • the supernatant was centrifuged at 100,000 ⁇ g for 200 minutes, and the precipitate was suspended in 10 mM HEPES buffer to obtain an EV sample.
  • the EV sample was introduced into the micro-channel of the analysis chip in which the channel for adjusting the liquid level was arranged in parallel, and the barycentric position of each particle was visualized and tracked by scattered light imaging.
  • the particle size was estimated by Brownian motion analysis, and the zeta potential was estimated by electrophoresis analysis.
  • Example 1 The number of HL60 cells, which are human acute myeloid leukemia, was counted using a blood count plate, and the initial number of cells was adjusted to 1 ⁇ 10 7 .
  • a medium for cell culture a RPMI medium (normal medium) supplemented with 10% fetal bovine serum (FBS) was used.
  • FBS fetal bovine serum
  • LMB-added medium obtained by adding 100 nM Leptomycin B (LMB), which is an agent for synchronizing cells to the G1 phase, in a normal medium was used as a medium for cell cycle synchronization.
  • a PRMI medium supplemented with 10% FBS from which EV was removed by ultracentrifugation was used as a medium that does not contain EV (EV removal medium).
  • HL60 cells were cultured in a normal medium or a medium supplemented with LMB for 24 hours, then replaced with an EV removal medium, and cultured for 2 hours. Thereafter, cells and cell supernatant were collected. For the collected cells, the cell cycle was examined by the amount of intracellular DNA. Specifically, the cells were fixed with ethanol, stored at 4 ° C. for 24 hours or more, and then washed with PBS. Thereafter, RNase A was added to degrade RNA and incubated at 37 ° C.
  • EV was purified from the collected culture supernatant by the following procedure. To remove large particles such as cell debris and microvesicles, centrifugation was performed at 300 ⁇ g for 10 minutes, 2,000 ⁇ g for 20 minutes, and 10,000 ⁇ g for 100 minutes. Next, the supernatant was centrifuged at 100,000 ⁇ g for 200 minutes, and the precipitate was suspended in 10 mM HEPES buffer.
  • the prepared EV sample was introduced into the flow path of the liquid level difference compensation type analysis chip, set in a high-precision single nanoparticle measurement system (see FIG. 19), and the particle size and zeta potential were measured (see FIG. 20). .
  • An outline of the procedure from cell culture to EV recovery is shown in FIG.
  • An outline of the procedure of the method for purifying EV from cells is shown in FIG.
  • FIG. 23 shows the result of FACS measurement of the fluorescence intensity of DNA stained with PI.
  • the cells cultured in the LMB-added medium are 36.4% to 14.5% in the S phase cells and 22.2 in the G2 / M phase, compared to the cells cultured in the normal medium (without the LMB medium). From 1% to 14.5%.
  • G1 phase cells increased from 41.4% to 69.7%.
  • LMB inhibited the transition from the G1 phase to the S phase, confirming that the cell cycle was synchronized with the G1 phase (FIG. 23).
  • the measurement results of EV particle size and zeta potential are shown in FIG.
  • the average particle size and standard deviation of EV particle size and zeta potential derived from cells cultured in normal medium were 129 ⁇ 80.3 nm and ⁇ 12.2 ⁇ 5.73 mV, respectively.
  • the average value and standard deviation of the particle size and zeta potential of EVs derived from cells cultured in the LMB-added medium were 193 ⁇ 115 nm and ⁇ 13.4 ⁇ 2.93 mV, respectively.
  • EV-derived cells cultured in a medium supplemented with LMB had a smaller zeta potential distribution range.
  • the distribution range (standard deviation) of the zeta potential of the EV secreted from the cells was reduced, and it was found that the EV secreted for each cell cycle was different. .
  • the standard deviation of the zeta potential is 2.93 mV, and the standard deviation of EV secreted from cells not synchronized in the cell cycle (5.73 mV) The standard deviation value was smaller. From this result, it was confirmed that the standard deviation of the zeta potential of EV can be reduced by synchronizing the cell cycle.
  • a method for producing an extracellular vesicle and a method for evaluating the quality of the extracellular vesicle such as a group of extracellular vesicles having a uniform quality and a composition containing the extracellular vesicle.
  • the extracellular vesicles of the present invention can be used for various uses such as pharmaceuticals, cosmetics, and foods.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Developmental Biology & Embryology (AREA)
  • Dispersion Chemistry (AREA)
  • Nutrition Science (AREA)
  • Virology (AREA)
  • Polymers & Plastics (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Provided is an extracellular-vesicle population in which the standard deviation of the zeta potential of extracellular vesicles is equal to or less than 5 mV. The present invention also provides a composition containing the extracellular-vesicle population. In addition, the present invention provides a method for manufacturing an extracellular-vesicle population, the method including: (a) a step for synchronizing cell cycles of a plurality of cells; (b) a step for exchanging, subsequent to the step (a), the medium for the plurality of cells with a medium that contains substantially no extracellular vesicles; (c) a step for culturing the plurality of cells in the medium that has been exchanged; and (d) a step for recovering the extracellular-vesicle population from the medium after the step (c).

Description

細胞外小胞の集団、及びその製造方法Extracellular vesicle population and method for producing the same
　本発明は、細胞外小胞の集団、及びその製造方法に関する。さらに、細胞外小胞の集団の品質を評価する方法、並びに細胞外小胞を含む組成物等に関する。
　本願は、２０１８年３月２日に、米国に出願された６２／６３７，３９７号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a population of extracellular vesicles and a method for producing the same. Furthermore, the present invention relates to a method for evaluating the quality of a population of extracellular vesicles, a composition containing extracellular vesicles, and the like.
This application claims priority based on 62 / 637,397 filed in the United States on March 2, 2018, the contents of which are incorporated herein by reference.
　エクソソームは、細胞が分泌する細胞外小胞の一種である。エクソソームは、がんをはじめとする難治疾患の早期検出や治療効果判定に利用できる疾病マーカー候補として注目されている。エクソソームは、さらに、薬物送達システム（ＤＤＳ）のキャリアとしての利用、及び間葉系幹細胞由来エクソソームによる再生医療等の治療応用への期待も高まりつつある。しかしながら、エクソソームのように、直径が数１０～１００ｎｍであって、かつ、不均質な粒子集団を分析・同定することは難しい。 Exosome is a kind of extracellular vesicle secreted by cells. Exosomes are attracting attention as candidate disease markers that can be used for early detection of intractable diseases including cancer and determination of therapeutic effects. Exosomes are also expected to be used as carriers for drug delivery systems (DDS) and therapeutic applications such as regenerative medicine using mesenchymal stem cell-derived exosomes. However, like exosomes, it is difficult to analyze and identify a heterogeneous particle population having a diameter of several tens to 100 nm and a heterogeneity.
　エクソソーム等の細胞外小胞を医薬品等として利用するためには、品質の均一性が問題となる。すなわち、医薬品等として製造・販売するためには、「医薬品及び医薬部外品の製造管理及び品質管理の基準」（Ｇｏｏｄ　Ｍａｎｕｆａｃｔｕｒｉｎｇ　Ｐｒａｃｔｉｃｅ；ＧＭＰ）に準じて製造する必要がある。
　しかしながら、細胞の培養上清から得られる細胞外小胞は、その性質が不均一な集団であり、品質の揃った細胞外小胞を得ることは難しい。またそれを評価する方法すら確立されていないのが現状である。 In order to use extracellular vesicles such as exosomes as pharmaceuticals, uniformity of quality becomes a problem. That is, in order to manufacture and sell as pharmaceuticals, etc., it is necessary to manufacture in accordance with “Manufacturing Standards and Quality Control Standards for Pharmaceuticals and Quasi-drugs” (Good Manufacturing Practice; GMP).
However, the extracellular vesicles obtained from the cell culture supernatant are a heterogeneous population, and it is difficult to obtain extracellular vesicles of uniform quality. In addition, there is no established method for evaluating it.
　本発明は、上記事情に鑑みてなされたものであり、品質の揃った細胞外小胞の集団、及び当該細胞外小胞を含む組成物等、当該細胞外小胞を製造する方法、並びに細胞外小胞の品質を評価する方法を提供することを課題とする。 The present invention has been made in view of the above circumstances, and a method for producing such extracellular vesicles, such as a group of extracellular vesicles of uniform quality and a composition containing such extracellular vesicles, and cells It is an object to provide a method for evaluating the quality of outer vesicles.
　本発明は、以下の態様を含む。
［１］細胞外小胞のゼータ電位の標準偏差が５ｍＶ以下である、細胞外小胞の集団。
［２］前記ゼータ電位の標準偏差が４．５ｍＶ以下である、［１］に記載の細胞外小胞の集団。
［３］前記ゼータ電位の標準偏差が４ｍＶ以下である、［２］に記載の細胞外小胞の集団。
［４］前記ゼータ電位の標準偏差が３．５ｍＶ以下である、［３］に記載の細胞外小胞の集団。
［５］前記ゼータ電位の標準偏差が３ｍＶ以下である、［４］に記載の細胞外小胞の集団。
［６］前記細胞外小胞がエクソソームである、［１］～［５］のいずれか一つに記載の細胞外小胞の集団。
［７］（ａ）複数の細胞の細胞周期を同調させる工程と、（ｂ）前記工程（ａ）後、前記複数の細胞の培地を、細胞外小胞を実質的に含まない培地に交換する工程と、
　（ｃ）前記培地交換した培地で、前記複数の細胞を培養する工程と、（ｄ）前記工程（ｃ）後の培地から、細胞外小胞の集団を回収する工程と、
　を含む、細胞外小胞の集団の製造方法。
［８］前記工程（ｄ）後に得られる細胞外小胞の集団が、［１］～［６］のいずれか一つに記載の細胞外小胞の集団である、［７］に記載の細胞外小胞の集団の製造方法。
［９］前記工程（ａ）を、細胞周期同調剤を含む培地で、前記複数の細胞を培養することにより行う、［７］又は［８］に記載の細胞外小胞の集団の製造方法。
［１０］前記工程（ａ）を、コンフルエントな状態で、前記複数の細胞を培養することにより行う、［７］又は［８］に記載の細胞外小胞の集団の製造方法。
［１１］前記工程（ａ）において、前記複数の細胞をＧ１期に同調させる、［７］～［１０］のいずれか一つに記載の細胞外小胞の集団の製造方法。
［１２］前記工程（ｄ）の後、さらに、（ｅ）前記の回収した細胞外小胞の集団に含まれる細胞外小胞のゼータ電位を測定する工程を含む、［７］～［１１］のいずれか一つに記載の細胞外小胞の集団の製造方法。
［１３］［７］～［１２］のいずれか一つに記載の細胞外小胞の集団の製造方法により製造された、細胞外小胞の集団。
［１４］細胞外小胞の集団の品質を評価する方法であって、（ａ）細胞外小胞の集団に含まれる複数の細胞外小胞のゼータ電位を測定する工程と、（ｂ）前記工程（ａ）で測定されたゼータ電位の標準偏差を算出する工程と、（ｃ）前記工程（ｂ）で算出された標準偏差に基づいて、前記細胞外小胞の集団の品質を評価する工程と、を含む、方法。
［１５］前記工程（ｃ）において、前記標準偏差が５ｍＶ以下である場合に、前記細胞外小胞の集団の均一性が高いと判定する、［１４］に記載の細胞外小胞の集団の品質を評価する方法。
［１６］前記工程（ｃ）において、前記標準偏差が４．５ｍＶ以下である場合に、前記細胞外小胞の集団の均一性が高いと判定する、［１５］に記載の細胞外小胞の集団の品質を評価する方法。
［１７］前記工程（ｃ）において、前記標準偏差が４ｍＶ以下である場合に、前記細胞外小胞の集団の均一性が高いと判定する、［１６］に記載の細胞外小胞の集団の品質を評価する方法。
［１８］前記工程（ｃ）において、前記標準偏差が３．５ｍＶ以下である場合に、前記細胞外小胞の集団の均一性が高いと判定する、［１７］に記載の細胞外小胞の集団の品質を評価する方法。
［１９］前記工程（ｃ）において、前記標準偏差が３ｍＶ以下である場合に、前記細胞外小胞の集団の均一性が高いと判定する、［１８］に記載の細胞外小胞の集団の品質を評価する方法。
［２０］複数の細胞外小胞を含む組成物であって、前記組成物に含まれる細胞外小胞のゼータ電位の標準偏差が５ｍＶ以下である、組成物。
［２１］前記ゼータ電位の標準偏差が４．５ｍＶ以下である、［２０］に記載の組成物。
［２２］前記ゼータ電位の標準偏差が４ｍＶ以下である、［２１］に記載の組成物。
［２３］前記ゼータ電位の標準偏差が３．５ｍＶ以下である、［２２］に記載の組成物。
［２４］前記ゼータ電位の標準偏差が３ｍＶ以下である、［２３］に記載の組成物。
［２５］前記細胞外小胞がエクソソームである、［２０］～［２４］のいずれか一つに記載の組成物。
［２６］前記組成物が医薬組成物、化粧品、又は食品である、［２０］～［２５］のいずれか一つに記載の組成物。
［２７］前記食品が、健康食品又は機能性食品である、［２６］に記載の組成物。
［２８］［１］～［６］のいずれか一つに記載の細胞外小胞の集団を含む、医薬組成物。
［２９］［１］～［６］のいずれか一つに記載の細胞外小胞の集団を含む、化粧品。
［３０］［１］～［６］のいずれか一つに記載の細胞外小胞の集団を含む、食品。
［３１］健康食品又は機能性食品である、［３０］に記載の食品 The present invention includes the following aspects.
[1] A population of extracellular vesicles having a standard deviation of zeta potential of extracellular vesicles of 5 mV or less.
[2] The population of extracellular vesicles according to [1], wherein the standard deviation of the zeta potential is 4.5 mV or less.
[3] The population of extracellular vesicles according to [2], wherein the standard deviation of the zeta potential is 4 mV or less.
[4] The population of extracellular vesicles according to [3], wherein the standard deviation of the zeta potential is 3.5 mV or less.
[5] The population of extracellular vesicles according to [4], wherein the standard deviation of the zeta potential is 3 mV or less.
[6] The population of extracellular vesicles according to any one of [1] to [5], wherein the extracellular vesicle is an exosome.
[7] (a) A step of synchronizing the cell cycle of a plurality of cells; (b) After the step (a), the medium of the plurality of cells is replaced with a medium substantially free of extracellular vesicles. Process,
(C) culturing the plurality of cells in the medium exchanged, (d) recovering a population of extracellular vesicles from the medium after step (c);
A method for producing a population of extracellular vesicles, comprising:
[8] The cell according to [7], wherein the population of extracellular vesicles obtained after the step (d) is the population of extracellular vesicles according to any one of [1] to [6]. A method for producing a population of outer vesicles.
[9] The method for producing a population of extracellular vesicles according to [7] or [8], wherein the step (a) is performed by culturing the plurality of cells in a medium containing a cell cycle synchronizer.
[10] The method for producing a population of extracellular vesicles according to [7] or [8], wherein the step (a) is performed by culturing the plurality of cells in a confluent state.
[11] The method for producing a population of extracellular vesicles according to any one of [7] to [10], wherein in the step (a), the plurality of cells are synchronized with G1 phase.
[12] After the step (d), the method further includes (e) a step of measuring the zeta potential of the extracellular vesicles contained in the collected extracellular vesicle population. [7] to [11] A method for producing a population of extracellular vesicles according to any one of the above.
[13] A population of extracellular vesicles produced by the method for producing a population of extracellular vesicles according to any one of [7] to [12].
[14] A method for evaluating the quality of a population of extracellular vesicles, wherein (a) measuring the zeta potential of a plurality of extracellular vesicles contained in the population of extracellular vesicles; Calculating the standard deviation of the zeta potential measured in step (a), and (c) evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b). And a method comprising:
[15] In the step (c), when the standard deviation is 5 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality.
[16] In the step (c), when the standard deviation is 4.5 mV or less, it is determined that the uniformity of the population of the extracellular vesicles is high. A method of assessing the quality of a population.
[17] In the step (c), when the standard deviation is 4 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality.
[18] In the step (c), when the standard deviation is 3.5 mV or less, it is determined that the uniformity of the population of extracellular vesicles is high. A method of assessing the quality of a population.
[19] In the step (c), when the standard deviation is 3 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality.
[20] A composition comprising a plurality of extracellular vesicles, wherein the standard deviation of the zeta potential of the extracellular vesicles contained in the composition is 5 mV or less.
[21] The composition according to [20], wherein the standard deviation of the zeta potential is 4.5 mV or less.
[22] The composition according to [21], wherein the standard deviation of the zeta potential is 4 mV or less.
[23] The composition according to [22], wherein the standard deviation of the zeta potential is 3.5 mV or less.
[24] The composition according to [23], wherein the standard deviation of the zeta potential is 3 mV or less.
[25] The composition according to any one of [20] to [24], wherein the extracellular vesicle is an exosome.
[26] The composition according to any one of [20] to [25], wherein the composition is a pharmaceutical composition, a cosmetic, or a food.
[27] The composition according to [26], wherein the food is a health food or a functional food.
[28] A pharmaceutical composition comprising the population of extracellular vesicles according to any one of [1] to [6].
[29] A cosmetic comprising the population of extracellular vesicles according to any one of [1] to [6].
[30] A food comprising the population of extracellular vesicles according to any one of [1] to [6].
[31] The food according to [30], which is a health food or a functional food
　本発明によれば、品質の揃った細胞外小胞の集団及び当該細胞外小胞を含む組成物等、当該細胞外小胞を製造する方法、並びに細胞外小胞の品質を評価する方法が提供される。 According to the present invention, there is provided a method for producing an extracellular vesicle and a method for evaluating the quality of the extracellular vesicle, such as a group of extracellular vesicles having a uniform quality and a composition containing the extracellular vesicle. Provided.
細胞外小胞のゼータ電位の測定に使用可能な粒子検出装置の概略的な平面図である。It is a schematic plan view of the particle | grain detection apparatus which can be used for the measurement of the zeta potential of an extracellular vesicle. 細胞外小胞のゼータ電位の測定に使用可能な粒子検出装置の概略的な正面図である。It is a schematic front view of the particle | grain detection apparatus which can be used for the measurement of the zeta potential of an extracellular vesicle. 細胞外小胞のゼータ電位の測定に使用可能な細胞外小胞分析チップの基本構造を示す斜視図である。It is a perspective view which shows the basic structure of the extracellular vesicle analysis chip | tip which can be used for the measurement of the zeta potential of an extracellular vesicle. 図３におけるＩＩ－ＩＩ線断面図である。It is the II-II sectional view taken on the line in FIG. ステージ部の設置面に流体デバイスが設置された平面図である。It is the top view in which the fluid device was installed in the installation surface of a stage part. 流体デバイスをｙｚ平面で部分的に断面した部分断面図である。FIG. 3 is a partial cross-sectional view of a fluid device partially cut along a yz plane. 図６におけるＡ－Ａ線断面図である。FIG. 7 is a cross-sectional view taken along line AA in FIG. 流体デバイスの照射部及び調整部の概略構成を示す図である。It is a figure which shows schematic structure of the irradiation part and adjustment part of a fluid device. 流体デバイスの調整部および流体デバイスの部分詳細図である。It is a partial detail drawing of the adjustment part of a fluid device, and a fluid device. 照明光がリザーバ部材の端面および流路の側面を通過する光路を模式的に示す図である。It is a figure which shows typically the optical path through which illumination light passes the end surface of a reservoir member, and the side surface of a flow path. 流体デバイスの制御装置の概略構成を示す図である。It is a figure which shows schematic structure of the control apparatus of a fluid device. 記憶部が記憶する粒子リストの一例を示す図である。It is a figure which shows an example of the particle | grain list | wrist which a memory | storage part memorize | stores. 記憶部が記憶する粒子相関リストの一例を示す図である。It is a figure which shows an example of the particle correlation list which a memory | storage part memorize | stores. 記憶部が記憶するしきい値の一例を示す図である。It is a figure which shows an example of the threshold value which a memory | storage part memorize | stores. 流体デバイスの制御装置の動作の一例を示す図である。It is a figure which shows an example of operation | movement of the control apparatus of a fluid device. 細胞周期を説明する図である。It is a figure explaining a cell cycle. 参考例１において、細胞外小胞のゼータ電位の多点計測を行った結果を示す図である。In the reference example 1, it is a figure which shows the result of having performed the multipoint measurement of the zeta potential of an extracellular vesicle. 参考例１において、細胞外小胞の粒径及びゼータ電位を測定した結果を示す図である。In Reference Example 1, it is a figure which shows the result of having measured the particle size and zeta potential of an extracellular vesicle. 実施例１で細胞外小胞の粒径を及びゼータ電位の計測に用いた、高精度単一ナノ粒子測定システムの構成の概略を示す図である。BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the outline of a structure of the highly accurate single nanoparticle measuring system used for the measurement of the particle size of an extracellular vesicle and zeta potential in Example 1. FIG. 粒径とゼータ電位の測定原理を説明する図である。It is a figure explaining the measurement principle of a particle size and zeta potential. 実施例１における、細胞培養から細胞外小胞（ＥＶ）を回収するまでの手順の概略を示す図である。It is a figure which shows the outline of the procedure in Example 1 until it collect | recovers an extracellular vesicle (EV) from cell culture. 実施例１における、細胞から細胞外小胞（ＥＶ）を精製する手順の概略を示す図である。BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the outline of the procedure in Example 1 which refine | purifies an extracellular vesicle (EV) from a cell. 実施例１で回収した細胞の細胞内ＤＮＡ量の分析結果を示す図である。It is a figure which shows the analysis result of the intracellular DNA amount of the cell collect | recovered in Example 1. FIG. 実施例１で回収した細胞外小胞の粒径及びゼータ電位の測定結果を示す図である。2 is a graph showing the measurement results of the particle size and zeta potential of extracellular vesicles collected in Example 1. FIG.
＜細胞外小胞の集団＞
　１実施形態において、本発明は、細胞外小胞のゼータ電位の標準偏差が５ｍＶ以下である、細胞外小胞の集団を提供する。 <Group of extracellular vesicles>
In one embodiment, the present invention provides a population of extracellular vesicles wherein the standard deviation of the zeta potential of the extracellular vesicle is 5 mV or less.
　細胞外小胞は、細胞が放出する小胞である。細胞外小胞の大きさは直径３０ｎｍから１μｍ程度である。細胞外小胞は、細胞の分泌物であり、その表面に分泌源の細胞由来のタンパク質を発現している。細胞外小胞の例としては、エクソソーム、アポトーシス小体、マイクロベシクル等が挙げられる。細胞外小胞の代表的なものとしては、エクソソームが挙げられる。エクソソームは、直径３０～２００ｎｍ程度の脂質小胞であり、エンドソームと細胞膜との融合体として、腫瘍細胞、樹状細胞、Ｔ細胞、Ｂ細胞等、種々の細胞から、血液、尿、唾液等の体液中に分泌される。
　生体内に存在する癌細胞等の異常細胞は、その細胞膜に特有のタンパク質を発現している。エクソソームは細胞の分泌物であり、その表面に分泌源の細胞由来のタンパク質を発現している。エクソソームの表面とは、細胞から分泌される脂質小胞の膜表面であって、分泌されたエクソソームが生体内の環境と接する部分をいう。
　細胞外小胞は、細胞が放出した細胞外小胞を加工したものであってもよい。細胞外小胞の加工方法は、加工後の細胞外小胞が、小胞構造を維持する限り、特に限定されない。細胞外小胞の加工方法としては、例えば、細胞外小胞の膜表面の修飾（例えば、ペプチドや糖鎖等による修飾）、細胞外小胞への薬剤の封入等が挙げられる。 Extracellular vesicles are vesicles released by cells. The size of the extracellular vesicle is about 30 nm to 1 μm in diameter. An extracellular vesicle is a secreted product of a cell and expresses a cell-derived protein as a secretory source on its surface. Examples of extracellular vesicles include exosomes, apoptotic bodies, microvesicles and the like. A typical example of an extracellular vesicle is exosome. Exosomes are lipid vesicles with a diameter of about 30 to 200 nm. As a fusion of endosomes and cell membranes, various cells such as tumor cells, dendritic cells, T cells, and B cells can be used for blood, urine, saliva, etc. Secreted into body fluids.
Abnormal cells such as cancer cells existing in the body express a protein specific to the cell membrane. An exosome is a secreted product of a cell and expresses a cell-derived protein as a secretory source on its surface. The surface of the exosome is a membrane surface of a lipid vesicle secreted from a cell, and refers to a portion where the secreted exosome is in contact with the environment in the living body.
Extracellular vesicles may be processed extracellular vesicles released by cells. The method for processing the extracellular vesicle is not particularly limited as long as the processed extracellular vesicle maintains the vesicle structure. Examples of extracellular vesicle processing methods include modification of the membrane surface of the extracellular vesicle (for example, modification with a peptide, sugar chain, etc.), encapsulation of a drug in the extracellular vesicle, and the like.
　「細胞外小胞の集団」とは、２個以上の細胞外小胞をいい、例えば、１０３個以上、１０４個以上、１０５個以上、１０６個以上の細胞外小胞が例示される。細胞外小胞の集団に含まれる細胞外小胞の個数の上限は特に限定されないが、例えば、１０１５個以下、１０１４個以下、１０１３個以下、１０１２個以下、１０１１個以下、又は１０１０個以下が例示される。細胞外小胞の集団は、例えば、１０３～１０１５個、１０４～１０１２以上、又は１０５～１０１０個の細胞外小胞であってもよい。 “A group of extracellular vesicles” refers to two or more extracellular vesicles, for example, 10 3 or more, 10 4 or more, 10 5 or more, 10 6 or more extracellular vesicles Is done. The upper limit of the number of extracellular vesicles contained in the population of extracellular vesicles is not particularly limited. For example, 10 15 or less, 10 14 or less, 10 13 or less, 10 12 or less, 10 11 or less, Or 10 10 or less are illustrated. The population of extracellular vesicles may be, for example, 10 3 to 10 15 , 10 4 to 10 12 or more, or 10 5 to 10 10 extracellular vesicles.
　本実施形態の細胞外小胞の集団は、同一種類の細胞を培養して得られた細胞外小胞の集団であることが好ましい。すなわち、本実施形態の細胞外小胞の集団を構成する細胞外小胞は、全て、同一種類の細胞が放出した細胞外小胞であることが好ましい。前記細胞は、細胞外小胞を放出する能力を有する限り、特に限定されない。そのような細胞としては、例えば、腫瘍細胞などの各種疾患細胞；樹状細胞、Ｔ細胞、Ｂ細胞などの免疫細胞；神経細胞などの各種組織細胞；脂肪細胞；間葉系幹細胞、造血幹細胞などの体性幹細胞；ＥＳ細胞、ｉＰＳ細胞などの多能性幹細胞；生殖細胞等が挙げられるが、これらに限定されない。前記細胞が由来する生物種は、特に限定されない。例えば、前記細胞は、ヒトの細胞であってもよい。また、ヒト以外の哺乳類、例えば、マウス、モルモット、サル、イヌ、ネコ、ウシ、ウマ、ブタ等の細胞であってもよい。細胞外小胞の集団は、例えば、腫瘍細胞などの各種疾患細胞；樹状細胞、Ｔ細胞、Ｂ細胞などの免疫細胞；神経細胞などの各種組織細胞；脂肪細胞；間葉系幹細胞、造血幹細胞などの体性幹細胞、ＥＳ細胞、ｉＰＳ細胞などの多能性幹細胞；生殖細胞等、種々の細胞から放出された細胞外小胞の集団であり得る。 The population of extracellular vesicles of this embodiment is preferably a population of extracellular vesicles obtained by culturing the same type of cells. That is, it is preferable that all the extracellular vesicles constituting the population of extracellular vesicles of this embodiment are extracellular vesicles released from the same type of cells. The cells are not particularly limited as long as they have the ability to release extracellular vesicles. Examples of such cells include various disease cells such as tumor cells; immune cells such as dendritic cells, T cells, and B cells; various tissue cells such as nerve cells; fat cells; mesenchymal stem cells, hematopoietic stem cells, and the like. Somatic stem cells; pluripotent stem cells such as ES cells and iPS cells; germ cells and the like, but not limited thereto. The biological species from which the cells are derived is not particularly limited. For example, the cell may be a human cell. Moreover, cells other than mammals other than humans, for example, mice, guinea pigs, monkeys, dogs, cats, cows, horses, pigs, etc. may be used. The extracellular vesicle population includes, for example, various disease cells such as tumor cells; immune cells such as dendritic cells, T cells and B cells; various tissue cells such as nerve cells; adipocytes; mesenchymal stem cells and hematopoietic stem cells. Pluripotent stem cells such as somatic stem cells such as ES cells, iPS cells, etc .; a population of extracellular vesicles released from various cells such as germ cells.
　本実施形態の細胞外小胞の集団は、細胞外小胞のゼータ電位の標準偏差が５ｍＶ以下である。ゼータ電位の標準偏差は、４．５ｍＶ以下であることが好ましく、４ｍＶ以下であることがより好ましく、３．５ｍＶ以下であることがさらに好ましく、３ｍＶ以下であることが特に好ましい。 The population of extracellular vesicles of this embodiment has a standard deviation of the zeta potential of the extracellular vesicles of 5 mV or less. The standard deviation of the zeta potential is preferably 4.5 mV or less, more preferably 4 mV or less, further preferably 3.5 mV or less, and particularly preferably 3 mV or less.
　前記ゼータ電位の標準偏差は、細胞外小胞の集団を構成する個々の細胞外小胞のゼータ電位を測定し、前記測定値から標準偏差を算出することにより求めることができる。ゼータ電位を測定する細胞外小胞の個数は、標準偏差の算出に十分な個数であればよく、細胞外小胞の集団の大きさ（細胞外小胞の個数）に応じて、適宜選択すればよい。例えば、ゼータ電位を測定する細胞外小胞の個数は、１００個以上、３００個以上、５００個以上、６００個以上、７００個以上、又は８００個以上等とすることができる。 The standard deviation of the zeta potential can be obtained by measuring the zeta potential of individual extracellular vesicles constituting the population of extracellular vesicles and calculating the standard deviation from the measured values. The number of extracellular vesicles for measuring the zeta potential is sufficient if it is sufficient to calculate the standard deviation, and is appropriately selected according to the size of the extracellular vesicle population (number of extracellular vesicles). That's fine. For example, the number of extracellular vesicles for measuring zeta potential can be 100 or more, 300 or more, 500 or more, 600 or more, 700 or more, or 800 or more.
　細胞外小胞の集団のゼータ電位の標準偏差は、下記式（ｓ）により算出することができる。 The standard deviation of the zeta potential of a population of extracellular vesicles can be calculated by the following formula (s).
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
　本実施形態の細胞外小胞の集団は、後述する細胞外小胞の集団の製造方法により、製造することができる。 The population of extracellular vesicles of this embodiment can be produced by a method for producing a population of extracellular vesicles described later.
　本実施形態の細胞外小胞の集団は、ゼータ電位の標準偏差が５ｍＶ以下であり、品質が揃った細胞外小胞の集団である。そのため、例えば、医薬品、化粧品、食品等の各種用途に利用可能である。本実施形態の細胞外小胞の集団は、品質が揃っているため、ドラッグデリバリーシステム（ＤＤＳ）のキャリアや再生医療等の医薬品としても好適に利用可能である。 The population of extracellular vesicles of this embodiment is a population of extracellular vesicles having a standard deviation of zeta potential of 5 mV or less and uniform quality. Therefore, it can be used for various uses such as pharmaceuticals, cosmetics, and foods. Since the population of extracellular vesicles of this embodiment has uniform quality, it can be suitably used as a drug delivery system (DDS) carrier or a pharmaceutical product such as regenerative medicine.
≪ゼータ電位の測定方法≫
　細胞外小胞の集団を構成する個々の細胞外小胞のゼータ電位の測定は、公知の方法、装置、又はシステムにより行うことができる。そのような方法、装置、又はシステムとしては、例えば、国際公開第２０１６／１７１１９８号、国際公開第２０１６／０６３９１２号、国際公開第２０１４／０３０５９０号等に記載の方法、装置、又はシステム等が例示される。以下に、細胞外小胞のゼータ電位測定装置の一例を記載する。 ≪Measurement method of zeta potential≫
The zeta potential of individual extracellular vesicles constituting the extracellular vesicle population can be measured by a known method, apparatus, or system. Examples of such a method, apparatus, or system include the method, apparatus, or system described in International Publication No. 2016/171198, International Publication No. 2016/063912, International Publication No. 2014/030590, and the like. Is done. Below, an example of the zeta potential measuring device of an extracellular vesicle is described.
　以下に記載する例では、細胞外小胞に特異的結合物質を結合させてゼータ電位の測定を行っているが、特異的結合物質を結合させることなく細胞外小胞のゼータ電位を測定することも可能である。本実施形態の細胞外小胞の集団のゼータ電位は、細胞外小胞に特異的結合物質を結合させないで測定を行なうことが好ましい。すなわち、以下に例示する説明において、特異的結合物質と細胞外小胞との結合反応は行わないことが好ましい。 In the example described below, the zeta potential is measured by binding a specific binding substance to the extracellular vesicle, but the zeta potential of the extracellular vesicle is measured without binding the specific binding substance. Is also possible. The zeta potential of the extracellular vesicle population of this embodiment is preferably measured without binding a specific binding substance to the extracellular vesicle. That is, in the explanation illustrated below, it is preferable not to perform the binding reaction between the specific binding substance and the extracellular vesicle.
　あるいは、特定の膜タンパク質を発現する細胞から放出された細胞外小胞である場合、当該タンパク質に対する特異的結合物質を用いて細胞外小胞のゼータ電位を測定してもよい。この場合、前記特異的結合物質と細胞外小胞との結合反応を行った後、細胞外小胞のゼータ電位を測定する。この方法により測定されたゼータ電位の標準偏差が５ｍＶ以下である細胞外小胞の集団は、特定の膜タンパク質の発現量について均一性の高い細胞外小胞の集団である。
　また、複数種類の膜タンパク質に対して、それぞれの膜タンパク質に特異的な特異的結合物質を結合させて、細胞外小胞のゼータ電位を測定してもよい。この方法により測定されたゼータ電位の標準偏差が５ｍＶ以下である細胞外小胞の集団は、前記複数種類の膜タンパク質の発現量について均一性の高い細胞外小胞の集団である。
　したがって、本明細書において、「細胞外小胞のゼータ電位」という用語は、細胞外小胞に特異的結合物質を結合させないで測定したゼータ電位、細胞外小胞に特定の膜タンパク質に対する特異的結合物質を結合させて測定したゼータ電位（特異的結合物質－細胞外小胞複合体のデータ電位）、及び細胞外小胞に複数種類の膜タンパク質に対する各特異的結結合物質を結合させて測定したゼータ電位（複数の特異的結合物質－細胞外小胞複合体のデータ電位）、を包含する。なお、前記複数種類の膜タンパク質は、２種類以上であれば、特に限定されず、例えば、２～５０種類、２～３０種類、２～２０種類、２～１０種類等が例示される。 Alternatively, in the case of an extracellular vesicle released from a cell expressing a specific membrane protein, the zeta potential of the extracellular vesicle may be measured using a specific binding substance for the protein. In this case, after the binding reaction between the specific binding substance and the extracellular vesicle is performed, the zeta potential of the extracellular vesicle is measured. A population of extracellular vesicles having a standard deviation of zeta potential measured by this method of 5 mV or less is a population of extracellular vesicles having high uniformity with respect to the expression level of a specific membrane protein.
Alternatively, the zeta potential of the extracellular vesicle may be measured by binding a specific binding substance specific to each membrane protein to a plurality of types of membrane proteins. A population of extracellular vesicles having a standard deviation of zeta potential measured by this method of 5 mV or less is a population of extracellular vesicles with high uniformity in the expression levels of the plurality of types of membrane proteins.
Accordingly, in this specification, the term “extracellular vesicle zeta potential” refers to the zeta potential measured without binding a specific binding substance to the extracellular vesicle, specific to a specific membrane protein in the extracellular vesicle. Measured by binding zeta potential (specific binding substance-data potential of extracellular vesicle complex) measured by binding the binding substance, and by binding each specific binding substance to multiple types of membrane proteins to the extracellular vesicle. Zeta potentials (data potentials of multiple specific binding substances-extracellular vesicle complexes). The plurality of types of membrane proteins are not particularly limited as long as they are two or more types, and examples thereof include 2 to 50 types, 2 to 30 types, 2 to 20 types, and 2 to 10 types.
　本明細書において、「特異的結合物質」とは、特定の分子（例えば、タンパク質）と特異的に結合する能力を有する物質を意味する。「特定の分子と特異的に結合する」とは、当該特定分子に対して高い結合親和性を有し、他の分子に対しては結合親和性が低いことを意味する。特異的結合物質は、結合対象の特定分子により異なっており、当該特定分子の種類に応じて種々選択可能である。特異的結合物質が結合対象とする特定分子としては、細胞外小胞の表面に存在する分子が挙げられ、例えば、抗原、膜タンパク質、核酸、糖鎖、糖脂質等が例示される。タンパク質に対する特異的結合物質としては、例えば、抗体（キメラ抗体、ヒト化抗体、修飾抗体、多価抗体、多重特異抗体、抗体断片などの改変抗体を含む）、アプタマー（核酸アプタマー、ペプチドアプタマーなど）、リガンド分子等が挙げられる。特異的結合物質としての抗体のクラスは特に限定されず、ＩｇＧ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＭ等のいずれの抗体クラスであってもよい。ＩｇＧとしては、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４等が挙げられる。ＩｇＡとしては、ＩｇＡ１、ＩｇＡ２等が挙げられる。ＩｇＭとしては、ＩｇＭ１、ＩｇＭ２等が挙げられる。抗体断片としては、ｓｃＦｖ、Ｆａｂ、Ｆ（ａｂ’）２、Ｆｖ等が挙げられる。リガンド分子としては、結合対象の特定分子がレセプタータンパク質である場合の、当該レセプタータンパク質のリガンド等が挙げられる。例えば、結合対象の特定分子がインターロイキンである場合、リガンド分子としてはＧタンパク質等が挙げられる。 In the present specification, the “specific binding substance” means a substance having an ability to specifically bind to a specific molecule (for example, a protein). The phrase “specifically binds to a specific molecule” means that it has a high binding affinity for the specific molecule and a low binding affinity for other molecules. The specific binding substance varies depending on the specific molecule to be bound, and can be variously selected according to the type of the specific molecule. Specific molecules to be bound by specific binding substances include molecules present on the surface of extracellular vesicles, and examples include antigens, membrane proteins, nucleic acids, sugar chains, glycolipids, and the like. Specific binding substances for proteins include, for example, antibodies (including chimeric antibodies, humanized antibodies, modified antibodies, multivalent antibodies, multispecific antibodies, modified antibodies such as antibody fragments), aptamers (nucleic acid aptamers, peptide aptamers, etc.) And ligand molecules. The class of the antibody as a specific binding substance is not particularly limited, and may be any antibody class such as IgG, IgA, IgD, IgE, and IgM. Examples of IgG include IgG1, IgG2, IgG3, and IgG4. Examples of IgA include IgA1 and IgA2. Examples of IgM include IgM1 and IgM2. Examples of antibody fragments include scFv, Fab, F (ab ') 2, Fv and the like. Examples of the ligand molecule include a ligand of the receptor protein when the specific molecule to be bound is a receptor protein. For example, when the specific molecule to be bound is interleukin, examples of the ligand molecule include G protein.
　特異的結合物質は、標識物質で標識されていてもよい。標識物質としては、例えば、ビオチン、アビジン、ストレプトアビジン、ニュートラビジン、グルタチオン－Ｓ－トランスフェラーゼ、グルタチオン、蛍光色素、ポリエチレングリコール、メリト酸等の電荷分子等が挙げられる。 The specific binding substance may be labeled with a labeling substance. Examples of labeling substances include biotin, avidin, streptavidin, neutravidin, glutathione-S-transferase, glutathione, fluorescent dyes, polyethylene glycol, charged molecules such as melittic acid, and the like.
［ゼータ電位の測定に用いる粒子検出装置の構成例］
　図１は、ゼータ電位の測定に使用可能な粒子検出装置１００の概略的な平面図である。図２は、粒子検出装置１００の概略的な正面図である。 [Configuration example of particle detector used for zeta potential measurement]
FIG. 1 is a schematic plan view of a particle detection apparatus 100 that can be used for zeta potential measurement. FIG. 2 is a schematic front view of the particle detection apparatus 100.
　粒子検出装置１００は、流体デバイスＣを検出対象として流体デバイスＣに照明光Ｌ１を照射し、流体デバイスＣからの散乱光Ｌ２を観察することにより、流体デバイスＣ内の粒子に関する情報を検出する。粒子検出装置１００は、光源部ＬＳ、照射部２０、調整部ＣＬ、ステージ部ＳＴ、検出部３０、送信部４０、および制御装置５を備えている。粒子検出装置１００および流体デバイスＣによって粒子検出システム１が構成される。 The particle detection apparatus 100 detects information related to particles in the fluid device C by irradiating the fluid device C with the illumination light L1 and observing the scattered light L2 from the fluid device C with the fluid device C as a detection target. The particle detection apparatus 100 includes a light source unit LS, an irradiation unit 20, an adjustment unit CL, a stage unit ST, a detection unit 30, a transmission unit 40, and a control device 5. A particle detection system 1 is configured by the particle detection apparatus 100 and the fluid device C.
　以下の説明においては、ステージ部ＳＴの設置面ＳＴａと直交する直交面（不図示）と直交する方向をｘ方向（ｘ軸；第３方向）、設置面ＳＴａと平行でｘ方向と直交する方向をｙ方向（ｙ軸）、ｘ方向およびｙ方向と直交する鉛直方向をｚ方向（ｚ軸；第２方向）として適宜説明する。 In the following description, the direction orthogonal to the orthogonal surface (not shown) orthogonal to the installation surface STa of the stage part ST is the x direction (x axis; third direction), and the direction parallel to the installation surface STa and orthogonal to the x direction. The y direction (y axis) and the vertical direction perpendicular to the x direction and the y direction are appropriately described as the z direction (z axis; second direction).
　まず、検出対象である流体デバイスＣについて説明する。
　本実施形態における流体デバイスＣは、一例として、細胞外小胞を分析する際に用いられる電気泳動分析チップである。以下に、細胞外小胞としてエクソソームを分析する場合を例として、細胞外小胞分析チップ（電気泳動分析チップ）について説明する。 First, the fluid device C that is a detection target will be described.
The fluid device C in this embodiment is an electrophoresis analysis chip used when analyzing an extracellular vesicle as an example. In the following, an extracellular vesicle analysis chip (electrophoresis analysis chip) will be described by taking an example of analyzing exosomes as extracellular vesicles.
　［エクソソームの分析］
　細胞外小胞分析チップを用いたエクソソームの分析は、一例として次のようにして行うことができる。まず、検出対象のエクソソームを精製する。次に、エクソソームと特異的結合物質とを接触させる。特異的結合物質としては、エクソソームの表面に存在する分子に特異的に結合することができる物質を選択する。次に、細胞外小胞分析チップを用いて、エクソソームのゼータ電位を計測し、分析を行う。本分析は、エクソソームに限らず、広く細胞外小胞一般の分析にも適用できる。また、エクソソームは、特異的結合物質との接触を行うことなく、分析に供してもよい。 [Analysis of exosomes]
As an example, analysis of exosomes using an extracellular vesicle analysis chip can be performed as follows. First, the exosome to be detected is purified. Next, the exosome is brought into contact with the specific binding substance. As the specific binding substance, a substance that can specifically bind to a molecule present on the surface of the exosome is selected. Next, the zeta potential of exosome is measured and analyzed using an extracellular vesicle analysis chip. This analysis can be applied not only to exosomes but also to analysis of extracellular vesicles in general. In addition, the exosome may be subjected to analysis without contact with a specific binding substance.
（エクソソームの精製）
　まず、エクソソームを含有する試料からエクソソームを精製する。試料としては、細胞培養液等が挙げられる。 (Purification of exosomes)
First, exosomes are purified from a sample containing exosomes. Examples of the sample include a cell culture solution.
　エクソソームを精製する方法としては、超遠心分離、限外ろ過、連続フロー電気泳動、クロマトグラフィー、μ－ＴＡＳ（Ｍｉｃｒｏ－Ｔｏｔａｌ　Ａｎａｌｙｓｉｓ　Ｓｙｓｔｅｍｓ）デバイスを使用する方法等が挙げられる。 Examples of the method for purifying exosomes include ultracentrifugation, ultrafiltration, continuous flow electrophoresis, chromatography, and a method using a μ-TAS (Micro-Total Analysis Systems) device.
（エクソソームと特異的結合物質との反応）
　次に、エクソソームと特異的結合物質とを接触させる。エクソソームの表面に検出対象の分子が存在した場合、特異的結合物質－エクソソーム複合体が形成される。特異的結合物質を適切に選択することにより、例えば、癌、肥満、糖尿病、神経変性疾患等の疾患に関連する異常を検出することができる。また、エクソソーム表面での検出対象の分子の発現量を分析することができる。例えば、膜表面にあるペプチドやタンパク質を人工的に発現させたエクソソームに対し、そのペプチドやタンパク質に対して特異的に結合する特異的結合物質を用いるなど、機能を改変したエクソソームを評価することもできる。 (Reaction between exosome and specific binding substance)
Next, the exosome is brought into contact with the specific binding substance. When the molecule to be detected is present on the surface of the exosome, a specific binding substance-exosome complex is formed. By appropriately selecting a specific binding substance, for example, an abnormality associated with a disease such as cancer, obesity, diabetes, or neurodegenerative disease can be detected. Moreover, the expression level of the molecule to be detected on the exosome surface can be analyzed. For example, it is possible to evaluate exosomes with altered functions, such as using a specific binding substance that specifically binds to the peptide or protein of the exosome that artificially expresses the peptide or protein on the membrane surface. it can.
（ゼータ電位の計測）
　一例として、特異的結合物質として抗体を使用した場合について説明する。エクソソームと抗体とを反応させた後、抗体と反応させたエクソソームのゼータ電位を計測する。ゼータ電位とは、溶液中の微粒子の表面電荷である。例えば、エクソソームが負に帯電しているのに対し、抗体は正に帯電している。このため、抗体－エクソソーム複合体のゼータ電位は、エクソソーム単独のゼータ電位と比較して正にシフトしている。したがって、抗体と反応させたエクソソームのゼータ電位を測定することによって、エクソソームの膜表面における抗原の発現を検出することができる。これは、抗体に限らず、正に帯電した他の特異的結合物質でも同様である。 (Measurement of zeta potential)
As an example, the case where an antibody is used as a specific binding substance will be described. After reacting the exosome with the antibody, the zeta potential of the exosome reacted with the antibody is measured. The zeta potential is the surface charge of the fine particles in the solution. For example, exosomes are negatively charged while antibodies are positively charged. For this reason, the zeta potential of the antibody-exosome complex is shifted positively compared to the zeta potential of the exosome alone. Therefore, by measuring the zeta potential of the exosome reacted with the antibody, the expression of the antigen on the exosome membrane surface can be detected. This is true not only for antibodies but also for other positively charged specific binding substances.
　エクソソームのゼータ電位ζは、一例として、細胞外小胞分析チップのマイクロ流路内で、エクソソームの電気泳動を行い、エクソソームの電気泳動速度Ｓを光学的に測定し、測定されたエクソソームの電気泳動速度Ｓに基づいて、以下の式（１）に示すスモルコフスキー（Ｓｍｏｌｕｃｈｏｗｓｋｉ）の式を用いて算出することができる。 As an example, the exosome zeta potential ζ is obtained by performing exosome electrophoresis in the microchannel of an extracellular vesicle analysis chip, optically measuring the exosome electrophoresis speed S, and measuring the measured exosome electrophoresis. Based on the speed S, it can be calculated using the Smolkovsky equation shown in the following equation (1).
Figure JPOXMLDOC01-appb-M000002
Figure JPOXMLDOC01-appb-M000002
　式（１）中、Ｕは測定対象のエクソソームの電気泳動移動度、ε及びηは、それぞれ、サンプル溶液の誘電率及び粘性係数である。また、電気泳動移動度Ｕは、電気泳動速度Ｓをマイクロ流路内の電界強度で除して算出することができる。 In Equation (1), U is the electrophoretic mobility of the exosome to be measured, and ε and η are the dielectric constant and viscosity coefficient of the sample solution, respectively. The electrophoretic mobility U can be calculated by dividing the electrophoretic velocity S by the electric field strength in the microchannel.
　エクソソームの電気泳動速度Ｓは、一例として、エクソソームを、細胞外小胞分析チップのマイクロ流路内で電気泳動し、一例として、レーザー光を、マイクロ流路内を流れるエクソソームに照射して、レイリー散乱光による粒子画像を取得することにより、測定することができる。レーザー光としては、一例として、波長４０５ｎｍ、強度１５０ｍＷのものが挙げられる。 As an example, the exosome electrophoresis speed S is obtained by electrophoresing an exosome in a microchannel of an extracellular vesicle analysis chip, and as an example, irradiating the exosome flowing in the microchannel with a Rayleigh Measurement can be performed by obtaining a particle image by scattered light. As an example of the laser beam, one having a wavelength of 405 nm and an intensity of 150 mW can be given.
（粒子径の計測）
　エクソソームの粒子径ｄは、一例として、細胞外小胞分析チップのマイクロ流路内で、エクソソームの電気泳動を行い、エクソソームの電気泳動速度Ｓを光学的に測定し、測定されたエクソソームの電気泳動速度Ｓに基づいて、以下の式（２）に示すアインシュタイン・ストークスの式を用いて算出することができる。 (Measurement of particle size)
As an example, the particle diameter d of the exosome is obtained by performing exosome electrophoresis in the microchannel of the extracellular vesicle analysis chip, optically measuring the exosome electrophoresis speed S, and measuring the exosome electrophoresis. Based on the speed S, it can be calculated using the Einstein-Stokes equation shown in the following equation (2).
Figure JPOXMLDOC01-appb-M000003
Figure JPOXMLDOC01-appb-M000003
　式（２）中、ｄはエクソソームの粒子径、ｋはボルツマン定数、Ｔは絶対温度、ηはサンプル溶液の粘性係数、Ｄは微粒子の拡散係数である。すなわち、エクソソームの粒子径ｄは、測定対象のエクソソームのブラウン運動の状態に基づいて、算出することができる。 In formula (2), d is the particle diameter of exosome, k is Boltzmann constant, T is absolute temperature, η is the viscosity coefficient of the sample solution, and D is the diffusion coefficient of the fine particles. That is, the particle diameter d of the exosome can be calculated based on the Brownian motion state of the exosome to be measured.
［細胞外小胞分析チップの基本構造］
　図３は、細胞外小胞分析チップの基本構造を示す斜視図である。図４は、図３におけるＩＩ－ＩＩ線断面図である。細胞外小胞分析チップＣＨは、第１リザーバ１１０と、第２リザーバ１２０と、第１リザーバ１１０と第２リザーバ１２０とを接続する泳動流路１５０と、基材１６０とを備えている。泳動流路１５０は、例えば、ミリ流路やマイクロ流路である。泳動流路１５０は、一例として、幅２００μｍ、高さ４００μｍ、長さ１０ｍｍ程度の大きさである。泳動流路１５０は、細胞外小胞、あるいは、細胞外小胞の表面に存在する生体分子に特異的に結合する特異的結合物質と細胞外小胞とが相互作用してなる、特異的結合物質－細胞外小胞複合体（一例として、抗体－エクソソーム複合体）を電気泳動するものである。 [Basic structure of extracellular vesicle analysis chip]
FIG. 3 is a perspective view showing the basic structure of the extracellular vesicle analysis chip. 4 is a cross-sectional view taken along line II-II in FIG. The extracellular vesicle analysis chip CH includes a first reservoir 110, a second reservoir 120, an electrophoresis channel 150 that connects the first reservoir 110 and the second reservoir 120, and a base material 160. The migration channel 150 is, for example, a millimeter channel or a micro channel. For example, the migration channel 150 has a width of about 200 μm, a height of 400 μm, and a length of about 10 mm. The electrophoresis channel 150 is a specific binding formed by interaction between an extracellular vesicle or a specific binding substance that specifically binds to a biomolecule existing on the surface of the extracellular vesicle and the extracellular vesicle. The substance-extracellular vesicle complex (for example, antibody-exosome complex) is electrophoresed.
　泳動流路１５０は、その一方の端部が第１リザーバ１１０と接続され、その他方の端部が第２リザーバ１２０と接続されている。また、第１リザーバ１１０及び第２リザーバ１２０は、基材１６０に設けられ、それぞれ電極１３０及び電極１４０を有している。例えば、電極１３０は第１リザーバ１１０の底部に設けられ、電極１４０は第２リザーバ１２０の底部に設けられている。図４に示すように、電極１３０及び電極１４０は、それぞれ泳動流路１５０の端部の近傍に設けられている。また、例えば、第１リザーバ１１０は検体（例、分析対象のエクソソーム）が導入され、第２リザーバ１２０は緩衝液が導入される。なお、その緩衝液は第１リザーバ１１０に導入されてもよい。 The electrophoresis channel 150 has one end connected to the first reservoir 110 and the other end connected to the second reservoir 120. Further, the first reservoir 110 and the second reservoir 120 are provided on the base material 160 and have an electrode 130 and an electrode 140, respectively. For example, the electrode 130 is provided at the bottom of the first reservoir 110, and the electrode 140 is provided at the bottom of the second reservoir 120. As shown in FIG. 4, the electrode 130 and the electrode 140 are each provided in the vicinity of the end of the migration channel 150. Further, for example, a sample (eg, exosome to be analyzed) is introduced into the first reservoir 110, and a buffer solution is introduced into the second reservoir 120. Note that the buffer solution may be introduced into the first reservoir 110.
　細胞外小胞分析チップＣＨは、細胞外小胞のゼータ電位を計測するのに好適である。以下に、細胞外小胞としてエクソソームを分析する場合を例として、細胞外小胞分析チップＣＨを用いた、エクソソームのゼータ電位の測定方法について説明する。 The extracellular vesicle analysis chip CH is suitable for measuring the zeta potential of extracellular vesicles. In the following, a method for measuring the zeta potential of exosomes using the extracellular vesicle analysis chip CH will be described, taking as an example the case of analyzing exosomes as extracellular vesicles.
　まず、分析対象のエクソソームを含む試料液が、第１リザーバ１１０に導入される。分析対象のエクソソームは、特異的結合物質と反応させたものであってもよい。エクソソームは例えば培養上清から取得したものであり、試料液は、例えば、リン酸緩衝液（Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ、ＰＢＳ）等の緩衝液にエクソソームが懸濁されたエクソソーム懸濁液である。次に、エクソソームを含む試料液が泳動流路１５０に導入される。一例として、シリンジを第２リザーバ１２０に接続して試料液を吸引することにより、エクソソームを泳動流路１５０に導入することができる。次に、緩衝液を、第１リザーバ１１０及び第２リザーバ１２０に入れる。後述する液位調整手段により、第１リザーバ１１０と第２リザーバ１２０との液位（液面高）を調整して揃え、泳動流路１５０に生じる静水圧流の発生を防ぎ、ゼータ電位測定の精度を向上させることが可能となる。続いて、制御部（例、後述の制御装置５、又はコンピュータなど）によって電極１３０及び１４０の間に電圧を印加し、エクソソームを電気泳動する。一例として、制御部は約５０Ｖ／ｃｍの電界強度の電圧を約１０秒間印加する。 First, a sample solution containing exosomes to be analyzed is introduced into the first reservoir 110. The exosome to be analyzed may have been reacted with a specific binding substance. The exosome is obtained from, for example, the culture supernatant, and the sample solution is an exosome suspension in which the exosome is suspended in a buffer solution such as a phosphate buffer (Phosphate Buffered Saline, PBS). Next, a sample solution containing exosomes is introduced into the migration channel 150. As an example, the exosome can be introduced into the electrophoresis channel 150 by connecting a syringe to the second reservoir 120 and sucking the sample solution. Next, the buffer solution is put into the first reservoir 110 and the second reservoir 120. By adjusting the liquid level (liquid level height) between the first reservoir 110 and the second reservoir 120 by using a liquid level adjusting means, which will be described later, it is possible to prevent the generation of a hydrostatic pressure flow that occurs in the migration channel 150 and to measure the zeta potential. The accuracy can be improved. Subsequently, a voltage is applied between the electrodes 130 and 140 by a control unit (for example, a control device 5 described later or a computer), and the exosome is electrophoresed. As an example, the control unit applies a voltage having an electric field strength of about 50 V / cm for about 10 seconds.
　電気泳動中に、泳動流路１５０にレーザー光を照射し、泳動流路１５０からの出射光であるエクソソームを介した散乱光を、対物レンズ等を用いて集光し、受光センサ（例、高感度カメラ）を用いて、エクソソーム又は特異的結合物質－エクソソーム複合体を撮影する。対物レンズの倍率は、一例として６０倍程度である。レーザーの波長及び強度は、一例として、波長４０５ｎｍ、強度１５０ｍＷである。 During electrophoresis, the electrophoresis channel 150 is irradiated with laser light, and the scattered light passing through the exosome, which is emitted from the electrophoresis channel 150, is collected using an objective lens or the like, and a light receiving sensor (eg, high A sensitivity camera is used to image exosomes or specific binding substance-exosome complexes. The magnification of the objective lens is about 60 times as an example. The wavelength and intensity of the laser are, for example, a wavelength of 405 nm and an intensity of 150 mW.
　細胞外小胞分析チップＣＨを用いることにより、エクソソーム又は特異的結合物質－エクソソーム複合体のゼータ電位の平均値だけでなく、エクソソーム又は特異的結合物質－エクソソーム複合体のゼータ電位を１粒子レベルで計測することができる。そのため、個々のエクソソーム又は特異的結合物質－エクソソーム複合体のゼータ電位から、エクソソームの集団におけるゼータ電位の標準偏差を求めることができる。以下、エクソソーム又は特異的結合物質－エクソソーム複合体を、単に「エクソソーム」と記載する場合がある。 By using the extracellular vesicle analysis chip CH, not only the average value of the zeta potential of the exosome or specific binding substance-exosome complex, but also the zeta potential of the exosome or specific binding substance-exosome complex at the level of one particle. It can be measured. Therefore, the standard deviation of the zeta potential in a population of exosomes can be determined from the zeta potential of individual exosomes or specific binding substance-exosome complexes. Hereinafter, the exosome or the specific binding substance-exosome complex may be simply referred to as “exosome”.
［流体デバイスＣの構造］
　図５は、ステージ部ＳＴの設置面ＳＴａに流体デバイスＣが設置された平面図である。図６は、流体デバイスＣをｙｚ平面で部分的に断面した部分断面図である。図７は、図６におけるＡ－Ａ線断面図である。 [Structure of fluidic device C]
FIG. 5 is a plan view in which the fluid device C is installed on the installation surface STa of the stage unit ST. FIG. 6 is a partial cross-sectional view in which the fluid device C is partially cut in the yz plane. 7 is a cross-sectional view taken along line AA in FIG.
　図５に示すように、流体デバイスＣは、平面視矩形状に形成されている。図６に示すように、流体デバイスＣは、ｚ方向に順次積み重ねられたリザーバ部材（第１基材）１０および底板（第２基材）１１を備えている。例えば、流体デバイスＣは、少なくともリザーバ部材１０、底板１１で構成された、積層構造（積層体）である。
　この場合、流体デバイスＣの積層構造は二層構造となっている。例えば、このような流体デバイスＣの積層構造は、リザーバ部材１０と、底板１１とを互いに貼りあわせて形成される。 As shown in FIG. 5, the fluid device C is formed in a rectangular shape in plan view. As shown in FIG. 6, the fluid device C includes a reservoir member (first base material) 10 and a bottom plate (second base material) 11 that are sequentially stacked in the z direction. For example, the fluid device C has a laminated structure (laminated body) composed of at least the reservoir member 10 and the bottom plate 11.
In this case, the laminated structure of the fluid device C has a two-layer structure. For example, such a laminated structure of the fluid device C is formed by bonding the reservoir member 10 and the bottom plate 11 to each other.
　リザーバ部材１０は、外力などによって少なくとも一方向に弾性変形可能な材料で形成される。リザーバ部材１０の材料には、一例として、エラストマーであり、シリコーンゴム、ＰＤＭＳ（ポリジメチルシロキサン）などが挙げられる。底板１２は、照明光Ｌ１の照射によって発生した散乱光Ｌ２が透過する材料で形成されている。底板１２は、一例として、ガラス材で形成されている。 The reservoir member 10 is formed of a material that can be elastically deformed in at least one direction by an external force or the like. Examples of the material of the reservoir member 10 include elastomers such as silicone rubber and PDMS (polydimethylsiloxane). The bottom plate 12 is made of a material through which scattered light L2 generated by irradiation with the illumination light L1 is transmitted. The bottom plate 12 is formed of a glass material as an example.
　流体デバイスＣは、長さ方向（ｙ方向）に配列された複数（図５では３つ）のレーン２を備えている。各レーン２は、第１リザーバ１２Ａ、第２リザーバ１２Ｂ、流路１３および電極１８Ａ、１８Ｂを備えている。第１リザーバ１２Ａ及び第２リザーバ１２Ｂは、ｙ方向に間隔をあけて配置されている。例えば、第１リザーバ１２Ａ及び第２リザーバ１２Ｂは、流路１３の流路方向に間隔をあけて配置されている。このように、複数のレーンが流路方向に（直列に）配列されていることによって、側方からの光の照射が容易となる。
　複数のレーンをレーンごとに順番に分析してもよく、また、複数の検出系によって同時に分析してもよい。なお、複数のレーン２は高さ方向（ｚ方向）に配列されていてもよい。
　この場合、溶液は長さ方向（ｘ方向）から注入されてもよく、ｙ方向から注入されてもよい。照射光源は例えば複数あって、それぞれの光源が対応する高さのレーン２を流れる微粒子を照射する。また、少なくとも一つの照射光源から照射方向を変えることでレーン２を流れる微粒子を照射してもよい。 The fluidic device C includes a plurality of (three in FIG. 5) lanes 2 arranged in the length direction (y direction). Each lane 2 includes a first reservoir 12A, a second reservoir 12B, a flow path 13, and electrodes 18A and 18B. The first reservoir 12A and the second reservoir 12B are arranged at an interval in the y direction. For example, the first reservoir 12 </ b> A and the second reservoir 12 </ b> B are arranged at an interval in the flow path direction of the flow path 13. In this way, the plurality of lanes are arranged in the flow path direction (in series), so that it is easy to irradiate light from the side.
A plurality of lanes may be analyzed in order for each lane, or may be analyzed simultaneously by a plurality of detection systems. The plurality of lanes 2 may be arranged in the height direction (z direction).
In this case, the solution may be injected from the length direction (x direction) or from the y direction. For example, there are a plurality of irradiation light sources, and each of the light sources irradiates fine particles flowing through the lane 2 having a corresponding height. Further, the fine particles flowing in the lane 2 may be irradiated by changing the irradiation direction from at least one irradiation light source.
　ここで、レーン２が複数ある場合には、対物レンズの移動により照明光の形状を調整することにより、各レーン２に照射される照明光を調整してもよい。また、レーン２が複数ある場合には、流体デバイスＣが載置されるステージの移動によって、複数レーン２のうち測定対象のレーン２を選択（切換え）する構成であってもよい。 Here, when there are a plurality of lanes 2, the illumination light applied to each lane 2 may be adjusted by adjusting the shape of the illumination light by moving the objective lens. Further, when there are a plurality of lanes 2, a configuration in which the measurement target lane 2 is selected (switched) from among the plurality of lanes 2 by moving a stage on which the fluid device C is placed may be employed.
　第１リザーバ１２Ａは、ｘｙ平面と平行な面での断面が円形状でｚ方向に延在する保持空間１４Ａと、保持空間１４Ａの＋ｚ側端部から＋ｚ方向に向かうに従って漸次拡径する漏斗状の導入部１５Ａとを備えている。保持空間１４Ａは、－ｚ側の端部が底板１１と対向して開口する。保持空間１４Ａは、流路１３と接続される。 The first reservoir 12A has a holding space 14A that has a circular cross section in a plane parallel to the xy plane and extends in the z direction, and a funnel shape that gradually increases in diameter from the + z side end of the holding space 14A toward the + z direction. The introduction part 15A is provided. The holding space 14A opens at the −z side end facing the bottom plate 11. The holding space 14 </ b> A is connected to the flow path 13.
　第２リザーバ１２Ｂは、ｘｙ平面と平行な面での断面が円形状でｚ方向に延在する保持空間１４Ｂと、保持空間１４Ｂの＋ｚ側端部から＋ｚ方向に向かうに従って漸次拡径する漏斗状の導入部１５Ｂとを備えている。保持空間１４Ｂは、－ｚ側の端部が底板１１と対向して開口する。保持空間１４Ｂは、流路１３と接続される。 The second reservoir 12B has a holding space 14B having a circular cross section in a plane parallel to the xy plane and extending in the z direction, and a funnel shape gradually increasing in diameter from the + z side end of the holding space 14B toward the + z direction. The introduction part 15B is provided. The holding space 14B has an end on the −z side facing the bottom plate 11 and opening. The holding space 14 </ b> B is connected to the flow path 13.
　流路１３は、電気泳動用流路（電気泳動のための流路）である。流路１３は、流体デバイスＣの長さ方向であるｙ方向に延在する。流路１３は、底板１１と対向する側の面に保持空間１４Ａと保持空間１４Ｂとを接続するように設けられている。流路１３は、図７に示すように、リザーバ部材１０に形成された溝部１０Ａと、底板１１の表面（第２面）１１ａとで囲まれた断面矩形に形成されている。溝部１０Ａは、ｘ方向に対向する側面（第１面）１６ａ、１６ｂと、底板１１の表面１１ａとｚ方向で対向する底面（第２面）１６ｃに囲まれて形成される。側面１６ａ、１６ｂ、底面１６ｃおよび溝部１０Ａを構成する表面１１ａは鏡面加工されている。第１面は、第１側面である側面１６ａと第２側面である側面１６ｂとを含む。側面１６ａと側面１６ｂとは、互いに向かい合っており、第１方向であるｘ方向に互いに離間している。 The flow path 13 is a flow path for electrophoresis (flow path for electrophoresis). The flow path 13 extends in the y direction, which is the length direction of the fluid device C. The channel 13 is provided on the surface facing the bottom plate 11 so as to connect the holding space 14A and the holding space 14B. As shown in FIG. 7, the flow path 13 is formed in a rectangular cross section surrounded by the groove 10 </ b> A formed in the reservoir member 10 and the surface (second surface) 11 a of the bottom plate 11. The groove portion 10A is formed to be surrounded by side surfaces (first surfaces) 16a and 16b facing in the x direction and a bottom surface (second surface) 16c facing the surface 11a of the bottom plate 11 in the z direction. The side surfaces 16a, 16b, the bottom surface 16c, and the surface 11a constituting the groove 10A are mirror-finished. The first surface includes a side surface 16a that is a first side surface and a side surface 16b that is a second side surface. The side surface 16a and the side surface 16b face each other and are separated from each other in the x direction which is the first direction.
　レーン２は、流体デバイスＣの幅方向である照明光Ｌ１の光軸方向（入射方向）について、中心よりも＋ｘ側の端面１７に近い側に偏って配置されている。レーン２は、入射する照明光Ｌ１の光軸方向である流体デバイスＣの幅方向（図５におけるｘ方向）について、中心よりも照明光Ｌ１の入射側の端面１７に近い側に偏って配置されている。端面１７は、ｙ方向に関して少なくともレーン２が設けられる範囲が鏡面加工されている。流路１３は、一例として、幅２００μｍ、高さ（溝部１０Ａの深さ）４００μｍ、長さ１０ｍｍ程度の大きさに形成されている。 The lane 2 is arranged so as to be biased toward the side closer to the end surface 17 on the + x side than the center with respect to the optical axis direction (incident direction) of the illumination light L1 that is the width direction of the fluid device C. The lane 2 is arranged so as to be biased toward the side closer to the end surface 17 on the incident side of the illumination light L1 than the center in the width direction (the x direction in FIG. 5) of the fluid device C that is the optical axis direction of the incident illumination light L1. ing. The end face 17 is mirror-finished in a range where at least the lane 2 is provided in the y direction. For example, the channel 13 is formed in a size of about 200 μm in width, about 400 μm in height (depth of the groove 10A), and about 10 mm in length.
　底板１１の表面１１ａには、保持空間１４Ａに臨んで電極１８Ａが設けられている。底板１１の表面１１ａには、保持空間１４Ｂに臨んで電極１８Ｂが設けられている。電極１８Ａ及び電極１８Ｂの素材としては、金、白金、カーボン等が挙げられる。図７に示すように、底板１１における照明光Ｌ１の入射側に位置する端面（第２端面）１９は、ｘ方向について、リザーバ部材１０の端面１７の位置よりも、照明光Ｌ１の入射側とは逆側である－ｘ側に離間している。 An electrode 18A is provided on the surface 11a of the bottom plate 11 so as to face the holding space 14A. An electrode 18B is provided on the surface 11a of the bottom plate 11 so as to face the holding space 14B. Examples of the material for the electrode 18A and the electrode 18B include gold, platinum, and carbon. As shown in FIG. 7, the end surface (second end surface) 19 located on the incident side of the illumination light L1 in the bottom plate 11 is closer to the incident side of the illumination light L1 than the position of the end surface 17 of the reservoir member 10 in the x direction. Are spaced apart on the opposite side, -x side.
　図１に戻り、光源部ＬＳは、粒子に対して悪影響を及ぼさない波長として、上述したように、一例として、波長４０５ｎｍ、強度１５０ｍＷでビーム径（ピーク値に対して１／ｅ２となる径）０．８ｍｍでｚ方向を偏向方位とするレーザー光を照明光Ｌ１として発光する。なお、照明光Ｌ１は、偏光（例えば、直線偏光など）であっても、無偏光であってもよいが、本実施形態では、垂直偏光を用い、レイリー散乱の指向性が無い構成を採る。
　照明光Ｌ１は、上述した直交面と交差する方向に延びる光軸に沿って流体デバイスＣに照射される。本実施形態では、照明光Ｌ１の光軸は、ｘ方向と平行である。本実施形態の照明光Ｌ１は、ｘ方向に延びる光軸に沿って流体デバイスＣに照射される。 Returning to FIG. 1, as described above, the light source unit LS has a wavelength that does not adversely affect the particles. As an example, the light source unit LS has a wavelength of 405 nm and an intensity of 150 mW. Laser light having a deflection direction in the z direction at 0.8 mm is emitted as illumination light L1. The illumination light L1 may be polarized light (for example, linearly polarized light) or non-polarized light. However, in the present embodiment, vertical polarization is used and there is no directivity of Rayleigh scattering.
The illumination light L1 is applied to the fluid device C along an optical axis extending in a direction intersecting the orthogonal plane described above. In the present embodiment, the optical axis of the illumination light L1 is parallel to the x direction. The illumination light L1 of the present embodiment is applied to the fluid device C along the optical axis extending in the x direction.
　図８は、照射部２０及び調整部ＣＬの概略構成を示す図である。照射部２０は、照明光Ｌ１の光軸に沿って順次配置されたλ／２板２１およびエキスパンダレンズ２２を備えている。なお、図１に示される光源部ＬＳおよび照射部２０は、照明光Ｌ１の光軸がｙ方向に延びているが、最終的に流体デバイスＣ（流路１３）を照射する照明光Ｌ１はｘ方向に沿った光軸であるため、図８に示す照明光Ｌ１は、光軸がｘ方向に沿うものとして図示している。 FIG. 8 is a diagram illustrating a schematic configuration of the irradiation unit 20 and the adjustment unit CL. The irradiation unit 20 includes a λ / 2 plate 21 and an expander lens 22 that are sequentially arranged along the optical axis of the illumination light L1. In the light source unit LS and the irradiation unit 20 shown in FIG. 1, the optical axis of the illumination light L1 extends in the y direction, but the illumination light L1 that finally irradiates the fluid device C (channel 13) is x. Since the optical axis is along the direction, the illumination light L1 shown in FIG. 8 is illustrated on the assumption that the optical axis is along the x direction.
　光源部ＬＳが発光した照明光Ｌ１は、λ／２板２１を透過することで偏光方位がｙ方向に回転する。なお、光源部ＬＳがｙ方向を偏向方位とする照明光Ｌ１を発光する場合にはλ／２板２１は不要である。エキスパンダレンズ２２は、対向するシリンドリカルレンズ２２Ａ、２２Ｂを備える。シリンドリカルレンズ２２Ａ、２２Ｂは、ｙ方向についてはパワーを有していないため、照明光Ｌ１はｙ方向の幅が一定である。照明光Ｌ１のｚ方向の幅は、シリンドリカルレンズ２２Ａ、２２Ｂの光軸方向の距離に応じて拡大または縮小する。エキスパンダレンズ２２は、照明光Ｌ１のｚ方向の幅を、一例として、２倍に拡大する。 The illumination light L1 emitted from the light source unit LS is transmitted through the λ / 2 plate 21 so that the polarization direction is rotated in the y direction. Note that the λ / 2 plate 21 is not necessary when the light source unit LS emits the illumination light L1 having the deflection direction in the y direction. The expander lens 22 includes cylindrical lenses 22A and 22B facing each other. Since the cylindrical lenses 22A and 22B have no power in the y direction, the illumination light L1 has a constant width in the y direction. The width of the illumination light L1 in the z direction is enlarged or reduced according to the distance in the optical axis direction of the cylindrical lenses 22A and 22B. The expander lens 22 enlarges the width of the illumination light L1 in the z direction as an example by a factor of two.
　調整部ＣＬは、エキスパンダレンズ２２でｚ方向の幅が拡大されて入射した照明光Ｌ１を調整する。調整部ＣＬは、光源部ＬＳと対物レンズ３１との間の光路に配置されている。また、調整部ＣＬは、λ／２板２１又はエキスパンダレンズ２２と対物レンズ３１との間の光路に配置されている。調整部ＣＬは駆動機構を備えていてもよく、調整部ＣＬが移動することで収光点を調整できてもよい。調整部ＣＬは例えばｘ方向に駆動可能である。
　この場合、流路１３の位置が異なるチップを用いた場合であっても、流路１３内に収光点が位置するように調整することが可能である。また、収光点と流路１３の中心とがほぼ一致するように調整してもよく、検出部の中心部と収光点とがほぼ一致するように調整してもよい。
　図９は、実施形態に係る調整部ＣＬおよび流体デバイスＣの部分詳細図である。調整部ＣＬは、一例として、シリンドリカルレンズで構成される。調整部ＣＬは、照明光Ｌ１のｚ方向の幅が流路１３の内部において最小となり、且つ、流路１３の照射光入射側の側面１６ａの位置における照明光Ｌ１の通過領域が側面１６ａ内に限定されるように収束する収束角度に調整している。調整部ＣＬは、照明光Ｌ１のｚ方向の幅が流路１３の内部において最小となり、且つ、流路１３の照射光入射側の側面１６ａの位置における照明光Ｌ１の照射領域が側面１６ａ内に集光するような収束角度に照明光Ｌ１を調整している。また、調整部ＣＬは、流路１３の照射光射出側の側面１６ｂの位置における照明光Ｌ１（照射光束）の通過領域が側面１６ｂ内に限定されるように収束する収束角度に調整している。
　調整部ＣＬは、流路１３の照射光射出側の側面１６ｂの位置における照明光Ｌ１（照射光束）の照射領域が側面１６ｂ内に集光するような収束角度に照明光Ｌ１を調整している。
　また、調整部ＣＬは、リザーバ部材１０の端面１７の位置における照明光Ｌ１の照射領域が端面１７内に収束する収束角度に調整している。さらに、調整部ＣＬは、照明光Ｌ１が流路１３内の検出領域において収束点が存在するような収束角に調整している。
　例えば、流路１３の検出領域において検出部３０の焦点深度外の照明光Ｌ１の照明光束は焦点深度内の照明光束よりも少なくなるような収束角を有する。なお、例えば、上述の直交面は、リザーバ部材１０の端面１７、流路１３の照射光入射側の側面１６ａ、又は流路１３の照射光射出側の側面１６ｂを含む。 The adjustment unit CL adjusts the incident illumination light L <b> 1 that has been expanded by the expander lens 22 in the width in the z direction. The adjustment unit CL is disposed in the optical path between the light source unit LS and the objective lens 31. The adjustment unit CL is disposed in the optical path between the λ / 2 plate 21 or the expander lens 22 and the objective lens 31. The adjustment unit CL may include a drive mechanism, and the light collection point may be adjusted by the movement of the adjustment unit CL. The adjustment unit CL can be driven in the x direction, for example.
In this case, even when a chip having a different position of the flow path 13 is used, it is possible to adjust so that the light collecting point is located in the flow path 13. Further, adjustment may be made so that the light collecting point and the center of the flow path 13 substantially coincide with each other, or adjustment may be made so that the center part of the detection unit and the light collecting point substantially coincide with each other.
FIG. 9 is a partial detailed view of the adjustment unit CL and the fluid device C according to the embodiment. For example, the adjustment unit CL is configured by a cylindrical lens. The adjustment portion CL has a minimum width in the z-direction of the illumination light L1 inside the flow path 13, and the passage region of the illumination light L1 at the position of the side face 16a on the irradiation light incident side of the flow path 13 is within the side face 16a. The convergence angle is adjusted so as to be limited. The adjustment portion CL has a minimum width in the z direction of the illumination light L1 inside the flow path 13, and the irradiation area of the illumination light L1 at the position of the side face 16a on the irradiation light incident side of the flow path 13 is within the side face 16a. The illumination light L1 is adjusted to a convergence angle that condenses light. Moreover, the adjustment part CL is adjusted to the convergence angle which converges so that the passage area | region of the illumination light L1 (irradiation light beam) in the position of the side surface 16b by the side of irradiation light emission of the flow path 13 may be limited in the side surface 16b. .
The adjusting unit CL adjusts the illumination light L1 to a convergence angle such that the irradiation region of the illumination light L1 (irradiation light beam) at the position of the side surface 16b on the irradiation light emission side of the flow path 13 is condensed in the side surface 16b. .
Further, the adjustment unit CL adjusts the convergence angle so that the irradiation region of the illumination light L1 at the position of the end surface 17 of the reservoir member 10 converges in the end surface 17. Further, the adjustment unit CL adjusts the convergence angle so that the illumination light L1 has a convergence point in the detection region in the flow path 13.
For example, in the detection region of the flow path 13, the illumination light beam of the illumination light L1 outside the focal depth of the detection unit 30 has a convergence angle that is smaller than the illumination light beam within the focal depth. For example, the above-described orthogonal surface includes the end surface 17 of the reservoir member 10, the side surface 16a on the irradiation light incident side of the flow path 13, or the side surface 16b on the irradiation light emission side of the flow path 13.
　ここで、照明光Ｌ１が光軸方向（ｘ方向）について、流路１３の中央（ｘ＝０とする）でｚ方向の幅が最小幅ω０となる場合、照明光Ｌ１の流路１３内の媒質での収束角をθ、照明光Ｌ１の波長をλ、位置ｘおよび収束角θでのｚ方向のビーム幅をω（ｘ、θ）、照明光Ｌ１のビームプロファイルファクタをＭ２、最小幅ω０となるｘ方向の位置から側面１６ａまでの距離をｘＬとすると、下記の式（３）、式（４）において、式（５）を満足する必要がある。 Here, when the illumination light L1 is in the optical axis direction (x direction) and the width in the z direction is the minimum width ω0 at the center of the flow path 13 (x = 0), the illumination light L1 in the flow path 13 The convergence angle in the medium is θ, the wavelength of the illumination light L1 is λ, the beam width in the z direction at the position x and the convergence angle θ is ω (x, θ), the beam profile factor of the illumination light L1 is M2, and the minimum width ω0. Assuming that the distance from the position in the x direction to the side surface 16a is xL, the following formula (3) and formula (4) must satisfy formula (5).
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000004
Figure JPOXMLDOC01-appb-M000005
Figure JPOXMLDOC01-appb-M000005
Figure JPOXMLDOC01-appb-M000006
Figure JPOXMLDOC01-appb-M000006
　従って、調整部ＣＬは、少なくとも式（３）～（５）を満足し、且つｘ＝ｘＬのときのビーム幅ω（ｘＬ、θ）が側面１６ａのｚ方向の長さよりも小さく、側面１６ａ内に収束する収束角θで照明光Ｌ１を収束させるように調整された光学特性を有するものが設置される。 Therefore, the adjustment unit CL satisfies at least the expressions (3) to (5), and the beam width ω (xL, θ) when x = xL is smaller than the length of the side surface 16a in the z direction, A light source having an optical characteristic adjusted so as to converge the illumination light L1 at a convergence angle θ that converges on the light beam is installed.
　照明光Ｌ１がガウシアン光である場合には、上記の式（３）～（５）に含まれるビーム幅ω（ｘ、θ）は、照明光Ｌ１の強度がピーク値に対して１／ｅ２となる幅で規定される。収束角θが式（１）～（３）を満足する場合でも、ピーク値に対して１／ｅ２以下となる強度の照明光Ｌ１がビーム幅ω（ｘＬ、θ）の外側で側面１６ａの位置に入射するため、収束角θを設定する際はピーク値に対して１／ｅ２以下となる強度の照明光Ｌ１のビーム幅も考慮する。 When the illumination light L1 is Gaussian light, the beam width ω (x, θ) included in the above equations (3) to (5) is 1 / e2 with respect to the peak value of the intensity of the illumination light L1. It is specified by the width. Even when the convergence angle θ satisfies the expressions (1) to (3), the illumination light L1 having an intensity that is 1 / e2 or less with respect to the peak value is outside the beam width ω (xL, θ). Therefore, when the convergence angle θ is set, the beam width of the illumination light L1 having an intensity that is 1 / e2 or less with respect to the peak value is also taken into consideration.
　また、検出部３０によって照明光Ｌ１の光軸方向（ｘ方向）について、流路１３の全域を検出領域とするには、流路１３の全体に亘って照明光Ｌ１の光束内に検出部３０の焦点深度ＤＯＦが入る必要がある。照明光Ｌ１の光束内に検出部３０の焦点深度ＤＯＦが入る照明光Ｌ１の光束内に検出部３０の焦点深度ＤＯＦが入るためには、リザーバ部材１０の端面１７および流路１３の側面１６ａの光軸に対する傾きも考慮する必要がある。図１０は、照明光Ｌ１がリザーバ部材１０の端面１７および流路１３の側面１６ａを通過する光路を模式的に示す図である。流路１３の幅全体に亘って照明光Ｌ１の光束内に検出部３０の焦点深度ＤＯＦ（図９参照）が入るためには、下記の式（６）を満足する必要がある。 Further, in order for the detection unit 30 to set the entire area of the flow path 13 as a detection region in the optical axis direction (x direction) of the illumination light L1, the detection section 30 is included in the light flux of the illumination light L1 over the entire flow path 13. It is necessary to enter a DOF of DOF. In order for the depth of focus DOF of the detection unit 30 to fall within the luminous flux of the illumination light L1, the end surface 17 of the reservoir member 10 and the side surface 16a of the flow path 13 have It is also necessary to consider the inclination with respect to the optical axis. FIG. 10 is a diagram schematically showing an optical path through which the illumination light L1 passes through the end face 17 of the reservoir member 10 and the side face 16a of the flow path 13. In order for the focal depth DOF (see FIG. 9) of the detection unit 30 to enter the light flux of the illumination light L1 over the entire width of the flow path 13, the following expression (6) needs to be satisfied.
Figure JPOXMLDOC01-appb-M000007
Figure JPOXMLDOC01-appb-M000007
　ここで、角度δ３は、焦点面Ｆから見た照明光軸の仰角であり、焦点面Ｆから反時計回り方向を正方向とする。一方で、界面での入射角・出射角、リザーバ部材１０の端面１７及び流路１３の側面１６ａのｙｚ平面に対する傾斜角、空気中・流路デバイスＣの材質中・流路中の照明光束の焦点面Ｆに対する仰角、および流路デバイスＣの外側の媒質・流路デバイスＣの材質・流路１３内の媒質の屈折率には以下の関係が成立している。 Here, the angle δ3 is the elevation angle of the illumination optical axis viewed from the focal plane F, and the counterclockwise direction from the focal plane F is the positive direction. On the other hand, the incident angle and the emission angle at the interface, the inclination angle of the end surface 17 of the reservoir member 10 and the side surface 16a of the flow path 13 with respect to the yz plane, the illumination light flux in the material of the air / flow path device C and in the flow path The following relationship is established between the elevation angle with respect to the focal plane F, the medium outside the channel device C, the material of the channel device C, and the refractive index of the medium in the channel 13.
　ｎ１ｓｉｎα１＝ｎ２ｓｉｎα２　　　
　ｎ２ｓｉｎα３＝ｎ３ｓｉｎα４
　α１＋β１＝δ１
　α２＋β１＝δ２
　α３＋β２＝δ２
　α４＋β２＝δ３ n1sinα1 = n2sinα2
n2sin α3 = n3sin α4
α1 + β1 = δ1
α2 + β1 = δ2
α3 + β2 = δ2
α4 + β2 = δ3
　ここで、
　α１：自由空間からリザーバ部材１０の端面１７への照明光Ｌ１の入射角
　α２：端面１７からリザーバ部材１０内の照明光Ｌ１の出射角
　α３：リザーバ部材１０内から流路１３の壁面１６ａへの照明光Ｌ１の入射角
　α４：壁面１６ａから流路１３内部への照明光Ｌ１の出射角
　β１：端面１７の傾斜角
　β２：壁面１６ａの傾斜角
　δ１：自由空間においての照明光Ｌ１の焦点面Ｆからの仰角
　δ２：リザーバ部材１０内においての照明光Ｌ１の焦点面Ｆからの仰角
　δ３：流路１３内においての照明光Ｌ１の焦点面Ｆからの仰角
　ｎ１：自由空間媒質の屈折率
　ｎ２：リザーバ部材１０材質の屈折率
　ｎ３：流路１３内の媒質の屈折率
であり、
　入射角・出射角：端面１７および壁面１６ａへの垂線からの角度
　傾斜角：焦点面Ｆの垂線からの角度
　仰角：焦点面Ｆからの角度
としている。また、符号は全て反時計回り方向を正とする。 here,
α1: Incident angle of illumination light L1 from the free space to the end surface 17 of the reservoir member 10 α2: Output angle of illumination light L1 in the reservoir member 10 from the end surface 17 α3: From inside the reservoir member 10 to the wall surface 16a of the flow path 13 Incident angle of illumination light L1 α4: emission angle of illumination light L1 from wall surface 16a into flow path 13 β1: tilt angle of end surface 17 β2: tilt angle of wall surface 16a δ1: focal plane F of illumination light L1 in free space From the focal plane F of the illumination light L1 in the reservoir member δ3: Elevation angle from the focal plane F of the illumination light L1 in the flow path 13 n1: Refractive index of the free space medium n2: Reservoir Refractive index of material of member 10 n3: Refractive index of medium in flow path 13
Incident angle / outgoing angle: angle from the perpendicular to the end face 17 and the wall surface 16a Tilt angle: angle from the perpendicular to the focal plane F elevation angle: angle from the focal plane F. All signs are positive in the counterclockwise direction.
　上記の式から流路１３における照明光Ｌ１の仰角δ３は、以下の式（７）で表される。 From the above equation, the elevation angle δ3 of the illumination light L1 in the flow path 13 is expressed by the following equation (7).
Figure JPOXMLDOC01-appb-M000008
Figure JPOXMLDOC01-appb-M000008
　従って、流路１３のｘ方向の幅全体に亘って、照明光Ｌ１の光束内に検出部３０の焦点深度ＤＯＦが入るためには、以下の式（８）を満足する必要がある。 Therefore, in order for the focal depth DOF of the detection unit 30 to enter the luminous flux of the illumination light L1 over the entire width of the flow path 13 in the x direction, it is necessary to satisfy the following formula (8).
Figure JPOXMLDOC01-appb-M000009
Figure JPOXMLDOC01-appb-M000009
　従って、リザーバ部材１０の端面１７および流路１３の壁面１６ａの傾斜角、照明光Ｌ１の仰角δ３は、自由空間媒質の屈折率ｎ１、リザーバ部材１０の材質の屈折率ｎ２および流路１３内の媒質の屈折率ｎ３に応じて、式（８）を満足するように、選択・製造・調整されている必要がある。 Therefore, the inclination angle of the end face 17 of the reservoir member 10 and the wall surface 16a of the flow path 13 and the elevation angle δ3 of the illumination light L1 are the refractive index n1 of the free space medium, the refractive index n2 of the material of the reservoir member 10 and the flow path 13 In accordance with the refractive index n3 of the medium, it is necessary to be selected, manufactured and adjusted so as to satisfy Expression (8).
　ステージ部ＳＴは、図２に示すステージ駆動部６０の駆動によって、ｘ方向、ｙ方向およびｚ方向に移動する。ステージ駆動部６０の駆動は、制御装置５によって制御される。
　図５に示すように、ステージ部ＳＴは、流体デバイスＣが設置される設置面ＳＴａを備える。設置面ＳＴａは、ｘｙ平面と平行の面である。設置面ＳＴａは、ｙ方向に間隔をあけて配置されている。設置面ＳＴａは、流路デバイスＣのレーン２が設けられていないｙ方向の両端部を－Ｚ側から支持する。流体デバイスＣは、レーン２が配される領域が検出部３０による－Ｚ側からの観察に支障を来すことなく設置面ＳＴａに支持される。また、流体デバイスＣにおけるレーン２に照射されるまでの照明光Ｌ１の光路にステージ部ＳＴが存在しないため、流体デバイスＣに入射する照明光Ｌ１の一部がステージ部ＳＴに入射して、後述する粒子検出に悪影響を及ぼすことを抑制できる。 The stage unit ST moves in the x direction, the y direction, and the z direction by driving the stage driving unit 60 shown in FIG. The driving of the stage driving unit 60 is controlled by the control device 5.
As shown in FIG. 5, the stage unit ST includes an installation surface STa on which the fluid device C is installed. The installation surface STa is a surface parallel to the xy plane. The installation surfaces STa are arranged at intervals in the y direction. The installation surface STa supports both ends in the y direction where the lane 2 of the flow channel device C is not provided from the −Z side. In the fluid device C, the region where the lane 2 is arranged is supported on the installation surface STa without hindering the observation from the −Z side by the detection unit 30. Further, since the stage part ST does not exist in the optical path of the illumination light L1 until the lane 2 in the fluid device C is irradiated, a part of the illumination light L1 incident on the fluid device C enters the stage part ST, which will be described later. Adversely affecting the particle detection.
　設置面ＳＴａには、固定ピン５１が突出して設けられている。固定ピン５１は、流体デバイスＣの長辺に当接する二つの固定ピン５１ａと、流体デバイスＣの短辺に当接する一つの固定ピン５１ｂとから構成される。固定ピン５１ａは流体デバイスＣのｙ方向の両側の近傍にそれぞれ配置される。固定ピン５１ｂは、＋ｙ側に位置する短辺に当接する。当該＋ｙ側に位置する固定ピン５１ａと固定ピン５１ｂとが配置された角部と対角に位置する角部には、押し付けコマ５２が設けられている。押し付けコマ５２は、ステージ部ＳＴに対して流体デバイスＣを対角方向に押し付ける。押し付けられた流体デバイスＣは、固定ピン５１ａ、５１ｂに当接することで、流路１３（レーン２）がｙ方向と平行になるように、ｘｙ方向に関してステージ部ＳＴに位置決めされた状態で固定される。 The fixing pin 51 protrudes from the installation surface STa. The fixing pin 51 includes two fixing pins 51 a that contact the long side of the fluid device C and one fixing pin 51 b that contacts the short side of the fluid device C. The fixing pins 51a are arranged in the vicinity of both sides of the fluid device C in the y direction. The fixing pin 51b contacts the short side located on the + y side. A pressing piece 52 is provided at a corner located opposite to the corner where the fixing pin 51a and the fixing pin 51b located on the + y side are arranged. The pressing piece 52 presses the fluid device C diagonally against the stage part ST. The pressed fluid device C is fixed in a state where the fluid device C is positioned on the stage portion ST in the xy direction so that the flow path 13 (lane 2) is parallel to the y direction by contacting the fixing pins 51a and 51b. The
　検出部３０は、対物レンズ３１、撮像部３２を備えている。対物レンズ３１は、ステージ部ＳＴおよび流体デバイスＣの－Ｚ側に配置されている。図９に示すように、対物レンズ３１は、検出軸３１ａがｘ方向について流路１３の中心を通る位置に配置される。検出軸３１ａは、照明光Ｌ１の光軸と直交する。撮像部３２は、一例として、ＥＭＣＣＤ（Electron Multiplying Charge Coupled Device）カメラを備えており、入射する光の画像を撮像する。撮像部３２は、対物レンズ３１を介して入射する側方散乱光の画像情報を取得する。
　送信部４０は、撮像部３２で撮像された画像情報を制御装置５へ送信する。 The detection unit 30 includes an objective lens 31 and an imaging unit 32. The objective lens 31 is disposed on the −Z side of the stage unit ST and the fluid device C. As shown in FIG. 9, the objective lens 31 is disposed at a position where the detection axis 31a passes through the center of the flow path 13 in the x direction. The detection axis 31a is orthogonal to the optical axis of the illumination light L1. The imaging unit 32 includes an EMCD (Electron Multiplying Charge Coupled Device) camera as an example, and captures an image of incident light. The imaging unit 32 acquires image information of side scattered light that enters through the objective lens 31.
The transmission unit 40 transmits the image information captured by the imaging unit 32 to the control device 5.
［粒子検出装置の動作］
　粒子検出装置１００の動作は、設置工程、導入工程、照射工程、検出工程を含む。
　設置工程は、流体デバイスＣをステージ部ＳＴの設置面ＳＴａに設置する工程である。
　具体的には、図５に示したように、押し付けコマ５２により流体デバイスＣを対角方向に押し付けることにより、流体デバイスＣは、固定ピン５１ａ、５１ｂに押し付けられ、流路１３（レーン２）がｙ方向と平行になるように、ステージ部ＳＴに位置決めされた状態で設置面ＳＴａに設置される。 [Operation of particle detector]
The operation of the particle detection apparatus 100 includes an installation process, an introduction process, an irradiation process, and a detection process.
The installation process is a process of installing the fluid device C on the installation surface STa of the stage part ST.
Specifically, as shown in FIG. 5, by pressing the fluid device C diagonally with the pressing piece 52, the fluid device C is pressed against the fixing pins 51a and 51b, and the flow path 13 (lane 2). Is placed on the installation surface STa in a state of being positioned on the stage portion ST so as to be parallel to the y direction.
　導入工程は、粒子を含む試料を流体デバイスＣの保持空間１４Ａ、１４Ｂおよび流路１３に導入する工程である。試料としては、一例として、リン酸緩衝液等の緩衝液（媒質）にエクソソームが懸濁されたエクソソーム懸濁液を用いることができる。
　試料が流路１３に導入されたら、制御装置５はステージ駆動部６０を駆動して、検出対象となるレーン２が照明光Ｌ１の光路および検出部３０の検出軸３１ａ上に位置させる。
　検出対象となるレーン２が検出位置に移動すると、制御装置５は、電源部ＢＴを制御して電極１８Ａ及び電極１８Ｂに電界を印加させ、エクソソームを流路１３に沿って電気泳動させる力を付与する。一例として、制御装置５は、約５０Ｖ／ｃｍの電界強度の電圧を約１０秒間印加する。エクソソームの移動方向は、ｙ方向と平行である。 The introducing step is a step of introducing a sample containing particles into the holding spaces 14A and 14B and the flow path 13 of the fluid device C. As an example, an exosome suspension in which exosomes are suspended in a buffer solution (medium) such as a phosphate buffer can be used as the sample.
When the sample is introduced into the flow path 13, the control device 5 drives the stage drive unit 60 so that the lane 2 to be detected is positioned on the optical path of the illumination light L 1 and the detection axis 31 a of the detection unit 30.
When the detection target lane 2 moves to the detection position, the control device 5 controls the power supply unit BT to apply an electric field to the electrodes 18A and 18B, and applies a force for causing the exosome to electrophores along the flow path 13. To do. As an example, the control device 5 applies a voltage having an electric field strength of about 50 V / cm for about 10 seconds. The moving direction of the exosome is parallel to the y direction.
　照射工程は、照明光Ｌ１をｘ方向と平行に流路デバイスＣの流路１３に照射する工程である。
　照明光Ｌ１を照射する照射部２０および調整部ＣＬは、ｙ方向の幅が一定で、上述した式（３）～式（８）を満足する収束角θでｚ方向に収束するシートビーム状の照明光Ｌ１を照射する。照明光Ｌ１の最小ビーム厚（ｚ方向のビーム幅）は、一例として、１０μｍである。照明光Ｌ１の最小ビーム厚（ｚ方向のビーム幅）方向は、図７および図９のｚ方向またはｚ方向と平行な方向である。照明光Ｌ１の最小ビーム厚（ｚ方向のビーム幅）方向は、入射面（端面１７および側面１６ａ）における照明光Ｌ１の光軸方向及び流路方向とは異なる方向であり、該光軸方向及び流路方向と直交する方向である。流路方向は、流路１３が延在する方向である。流路方向は、流路１３を流体が流れる方向である。
　照射された照明光Ｌ１は、流体デバイスＣの一方の端面（照明光入射側端面）１７、流路１３の側面（照明光入射側側面）１６ａ、流路１３の内部、流路１３の側面（照明光射出側側面）１６ｂ、流体デバイスＣの他方の端面（照明光射出側端面）２７（図５参照）を順次通過する。照明光Ｌ１は、エクソソームの移動方向と直交する方向に照射される。
　照射された照明光Ｌ１は、図９に示すように、流路１３の内部においてｚ方向の幅が最小となるように収束し、且つ、流路１３の側面１６ａの位置における照射光束の通過領域が側面１６ａ内に限定されるように収束する。さらに、照射された照明光Ｌ１は、流路１３の照明光射出側の側面１６ｂの位置における照射光束の通過領域が側面１６ａ内に限定されるように収束する。照明光Ｌ１は、側面１６ａの位置における照射領域が側面１６ａ内に集光し、側面１６ｂの位置における照射領域が側面１６ｂ内に集光するような収束角に調整されている。また、照射された照明光Ｌ１は、流路１３における検出部３０の検出領域において収束点が存在する。 The irradiation process is a process of irradiating the flow path 13 of the flow path device C with the illumination light L1 parallel to the x direction.
The irradiation unit 20 and the adjustment unit CL that irradiate the illumination light L1 have a constant width in the y direction, and have a sheet beam shape that converges in the z direction at a convergence angle θ that satisfies the above-described equations (3) to (8). Irradiation light L1 is irradiated. As an example, the minimum beam thickness (beam width in the z direction) of the illumination light L1 is 10 μm. The minimum beam thickness (beam width in the z direction) direction of the illumination light L1 is the z direction in FIGS. 7 and 9 or a direction parallel to the z direction. The direction of the minimum beam thickness (beam width in the z direction) of the illumination light L1 is different from the optical axis direction and the flow path direction of the illumination light L1 on the incident surface (end surface 17 and side surface 16a). It is a direction orthogonal to the flow path direction. The channel direction is a direction in which the channel 13 extends. The flow path direction is a direction in which fluid flows through the flow path 13.
The irradiated illumination light L1 is one end surface (illumination light incident side end surface) 17 of the fluid device C, the side surface (illumination light incident side side surface) 16a of the flow channel 13, the inside of the flow channel 13, the side surface of the flow channel 13 ( The light passes through the illumination light emission side surface 16b and the other end surface (illumination light emission side end surface) 27 (see FIG. 5) of the fluid device C sequentially. The illumination light L1 is irradiated in a direction orthogonal to the moving direction of the exosome.
As shown in FIG. 9, the irradiated illumination light L1 converges so that the width in the z direction is minimized inside the flow path 13, and the irradiation light flux passage region at the position of the side surface 16 a of the flow path 13. Converges to be confined within the side surface 16a. Furthermore, the irradiated illumination light L1 converges so that the passage region of the irradiated light beam at the position of the side surface 16b on the illumination light exit side of the flow path 13 is limited to the side surface 16a. The illumination light L1 is adjusted to a convergence angle such that the irradiation region at the position of the side surface 16a is condensed in the side surface 16a and the irradiation region at the position of the side surface 16b is condensed in the side surface 16b. Further, the irradiated illumination light L1 has a convergence point in the detection region of the detection unit 30 in the flow path 13.
　検出工程は、照明光Ｌ１のｘ方向と平行な照射によって流路１３内部の粒子から生じる散乱光を検出部３０によって観察（イメージング）し検出する。検出部３０における対物レンズ３１の検出軸３１ａが照明光Ｌ１の光軸と直交しているため、検出部３０は粒子から生じる側方散乱光を検出する。検出部３０は、ｘ方向と平行に照射された照明光Ｌ１の照射によって、ｘ方向と垂直なｚ方向に向かって散乱した光を検出する。散乱光が観察された粒子の像は撮像部３２で撮像される。送信部４０は、撮像部３２で撮像された画像情報を制御装置５へ送信する。 In the detection step, the detection unit 30 observes (images) and detects the scattered light generated from the particles in the flow path 13 by irradiation of the illumination light L1 in parallel with the x direction. Since the detection axis 31a of the objective lens 31 in the detection unit 30 is orthogonal to the optical axis of the illumination light L1, the detection unit 30 detects side scattered light generated from the particles. The detection unit 30 detects light scattered toward the z direction perpendicular to the x direction by irradiation of the illumination light L1 irradiated in parallel with the x direction. The image of the particles in which the scattered light is observed is picked up by the image pickup unit 32. The transmission unit 40 transmits the image information captured by the imaging unit 32 to the control device 5.
［制御装置の構成］
　制御装置５は、粒子検出システム１を統括的に制御する。制御装置５は、ステージ駆動部６０を介してステージ部ＳＴおよび流体デバイスＣの移動を制御する。制御装置５は、電源部（印加部）ＢＴを制御して、電極１８Ａ、１８Ｂに流路１３に沿った方向の電界を印加させる。また、制御装置５は、粒子検出装置１００が撮像した画像を処理することにより、種々の判定を行う。この制御装置５の構成の詳細について、図１１から図１６を参照して説明する。 [Configuration of control device]
The control device 5 comprehensively controls the particle detection system 1. The control device 5 controls the movement of the stage unit ST and the fluid device C via the stage driving unit 60. The control device 5 controls the power supply unit (application unit) BT to apply an electric field in the direction along the flow path 13 to the electrodes 18A and 18B. Moreover, the control apparatus 5 performs various determinations by processing the image captured by the particle detection apparatus 100. Details of the configuration of the control device 5 will be described with reference to FIGS. 11 to 16.
　図１１は、本実施形態の制御装置５の概略構成を示す図である。制御装置５は、演算部５００と、記憶部５２０とを備えている。この記憶部５２０は、フラッシュメモリ、ＨＤＤ（Ｈａｒｄ　Ｄｉｓｋ　Ｄｒｉｖｅ）、ＲＡＭ（Ｒａｎｄｏｍ　Ａｃｃｅｓｓ　Ｍｅｍｏｒｙ）、ＲＯＭ（Ｒｅａｄ　Ｏｎｌｙ　Ｍｅｍｏｒｙ）、レジスタなどの記憶装置を備えている。記憶部５２０には、演算部５００が実行するプログラム（ファームウェア）が予め格納される。また、記憶部５２０には、演算部５００が演算処理を行った演算結果が格納される。 FIG. 11 is a diagram showing a schematic configuration of the control device 5 of the present embodiment. The control device 5 includes a calculation unit 500 and a storage unit 520. The storage unit 520 includes storage devices such as a flash memory, an HDD (Hard Disk Drive), a RAM (Random Access Memory), a ROM (Read Only Memory), and a register. The storage unit 520 stores a program (firmware) executed by the calculation unit 500 in advance. In addition, the storage unit 520 stores a calculation result obtained by performing the calculation process by the calculation unit 500.
　演算部５００は、ＣＰＵ（Ｃｅｎｔｒａｌ　Ｐｒｏｃｅｓｓｉｎｇ　Ｕｎｉｔ）を備えており、各種の演算を行う。この演算部５００は、その機能部として、取得部５０１と、識別部５０２と、ゼータ電位判定部５０３と、粒子径判定部５０４と、相関部５０５と、状態判定部５０６と、評価部５０７とを備えている。 The calculation unit 500 includes a CPU (Central Processing Unit) and performs various calculations. The calculation unit 500 includes, as its functional units, an acquisition unit 501, an identification unit 502, a zeta potential determination unit 503, a particle diameter determination unit 504, a correlation unit 505, a state determination unit 506, and an evaluation unit 507. It has.
　取得部５０１は、粒子検出装置１００が撮像した画像を取得する。具体的には、上述したように、粒子検出装置１００の撮像部３２は、対物レンズ３１を介して入射する側方散乱光の画像を撮像して、撮像した画像の画像情報を、送信部４０に出力する。取得部５０１は、撮像部３２が撮像した側方散乱光の画像の画像情報を、送信部４０を介して取得する。取得部５０１は、取得した画像を識別部５０２に出力する。 The acquisition unit 501 acquires an image captured by the particle detection device 100. Specifically, as described above, the imaging unit 32 of the particle detection apparatus 100 captures an image of side scattered light that is incident through the objective lens 31 and transmits image information of the captured image to the transmission unit 40. Output to. The acquisition unit 501 acquires the image information of the side scattered light image captured by the imaging unit 32 via the transmission unit 40. The acquisition unit 501 outputs the acquired image to the identification unit 502.
　識別部５０２は、粒子検出装置１００が撮像した画像のなかから、微粒子の画像を抽出する。例えば、識別部５０２は、取得部５０１から供給される画像に対して既知のフィルター処理やパターンマッチング処理を施すことにより、微粒子の画像を抽出する。このとき、識別部５０２は、抽出した微粒子の画像に対して、微粒子ごとに粒子番号を付与してもよい。なお、識別対象の微粒子が細胞外小胞である場合には、この粒子番号とは、細胞外小胞識別子であってもよい。つまり、識別部５０２は、微粒子の粒子に対してラベリングを行ってもよい。このことによって後述する相関部において、微粒子のゼータ電位ζと微粒子の粒子径ｄとの関連付けが容易となる。このラベリングの際には、取得部が取得した複数の画像のうち、第１の時刻において撮像された画像に含まれる第１の微粒子の画像と、第１の時刻とは異なる第２の時刻において撮像された画像に含まれる第２の微粒子の画像とが、同一の微粒子を示す画像であるか否かを、媒質中のブラウン運動による微粒子の移動量に基づいて判定してもよい。また、識別部５０２は、ラベリングした微粒子の粒子について、粒子検出装置１００が撮像した画像のフレーム間の差分に基づいて、トラッキングを行う。ここで、トラッキングとは、画像内の粒子の座標の経時的変化を追跡することをいう。識別部５０２が、微粒子のトラッキングを行った結果の一例を図１２に示す。 The identification unit 502 extracts a fine particle image from the image captured by the particle detection device 100. For example, the identification unit 502 extracts a fine particle image by performing known filter processing and pattern matching processing on the image supplied from the acquisition unit 501. At this time, the identification unit 502 may assign a particle number for each fine particle to the extracted fine particle image. When the identification target microparticle is an extracellular vesicle, the particle number may be an extracellular vesicle identifier. That is, the identification unit 502 may label the fine particles. This facilitates the correlation between the zeta potential ζ of the fine particles and the particle diameter d of the fine particles in the correlation section described later. During the labeling, among the plurality of images acquired by the acquisition unit, the first fine particle image included in the image captured at the first time and the second time different from the first time Whether or not the image of the second fine particle included in the captured image is an image showing the same fine particle may be determined based on the movement amount of the fine particle due to Brownian motion in the medium. Further, the identification unit 502 performs tracking on the labeled fine particle based on the difference between frames of the image captured by the particle detection device 100. Here, tracking refers to tracking changes with time in the coordinates of particles in an image. An example of the result of the identification unit 502 tracking fine particles is shown in FIG.
　図１２は、記憶部５２０が記憶する粒子リストＬＳ１の一例を示す図である。この粒子リストＬＳ１には、行方向をラベリングされた粒子番号とし、列方向を撮像時刻として、各時刻における各微粒子の画像の座標（Ｘ、Ｙ）が記憶される。この一例においては、時刻ｔ０から、時刻ｔ５０までの各時刻における、微粒子Ｐ１から微粒子Ｐｎまでの、各微粒子の座標が粒子リストＬＳ１に記憶される。 FIG. 12 is a diagram illustrating an example of the particle list LS1 stored in the storage unit 520. The particle list LS1 stores the coordinates (X, Y) of the image of each fine particle at each time, with the row direction as the labeled particle number and the column direction as the imaging time. In this example, the coordinates of each fine particle from the fine particle P1 to the fine particle Pn at each time from the time t0 to the time t50 are stored in the particle list LS1.
　図１１に戻り、ゼータ電位判定部５０３は、識別部５０２がトラッキングした結果に基づいて、微粒子毎のゼータ電位ζを判定する。例えば、ゼータ電位判定部５０３は、識別部５０２が行った微粒子Ｐ１についてのトラッキング結果のうち、時刻ｔ０から、時刻ｔ１までの微粒子Ｐ１の移動速度ｖ１に基づいて、微粒子Ｐ１のゼータ電位ζ１を判定する。
　ゼータ電位判定部５０３は、上述の式（１）に基づいてゼータ電位ζを判定する。なお、この一例では、サンプル溶液の誘電率ε及びサンプル溶液の粘性係数ηは、予め記憶部５２０に記憶されている。ゼータ電位判定部５０３は、記憶部５２０に記憶されているサンプル溶液の誘電率ε及びサンプル溶液の粘性係数ηと、識別部５０２によるトラッキング結果から求めた微粒子の移動速度とに基づいて、微粒子のゼータ電位ζを判定する。 Returning to FIG. 11, the zeta potential determination unit 503 determines the zeta potential ζ for each fine particle based on the result tracked by the identification unit 502. For example, the zeta potential determination unit 503 determines the zeta potential ζ1 of the fine particle P1 based on the moving speed v1 of the fine particle P1 from the time t0 to the time t1 in the tracking result of the fine particle P1 performed by the identification unit 502. To do.
The zeta potential determination unit 503 determines the zeta potential ζ based on the above equation (1). In this example, the dielectric constant ε of the sample solution and the viscosity coefficient η of the sample solution are stored in the storage unit 520 in advance. The zeta potential determination unit 503 is configured based on the permittivity ε of the sample solution and the viscosity coefficient η of the sample solution stored in the storage unit 520 and the moving speed of the particles obtained from the tracking result by the identification unit 502. The zeta potential ζ is determined.
　粒子径判定部５０４は、サンプル溶液中のブラウン運動による微粒子の移動量と、上述の式（２）とに基づいて、微粒子の径を判定する。ここでは、粒子径判定部５０４が、微粒子Ｐ１の粒子径を判定する場合の具体例について説明する。なお、この一例においては、ボルツマン定数ｋ及びサンプル溶液の絶対温度Ｔは、予め記憶部５２０に記憶されている。粒子径判定部５０４は、識別部５０２がトラッキングした結果に基づいて、微粒子Ｐ１の移動量を算出する。また、粒子径判定部５０４は、算出した微粒子Ｐ１の移動量と、記憶部５２０に記憶されているボルツマン定数ｋ及び絶対温度Ｔと、上述の式（２）とに基づいて、微粒子Ｐ１の粒子径ｄ１を判定する。 The particle diameter determination unit 504 determines the diameter of the fine particles based on the amount of movement of the fine particles due to Brownian motion in the sample solution and the above equation (2). Here, a specific example in which the particle size determination unit 504 determines the particle size of the fine particles P1 will be described. In this example, the Boltzmann constant k and the absolute temperature T of the sample solution are stored in the storage unit 520 in advance. The particle diameter determination unit 504 calculates the movement amount of the fine particles P1 based on the result tracked by the identification unit 502. In addition, the particle size determination unit 504 determines the particles of the fine particles P1 based on the calculated movement amount of the fine particles P1, the Boltzmann constant k and the absolute temperature T stored in the storage unit 520, and the above equation (2). The diameter d1 is determined.
　相関部５０５は、ゼータ電位判定部５０３が判定した微粒子のゼータ電位ζと、粒子径判定部５０４が判定した微粒子の粒子径ｄとを関連付ける。具体的には、ゼータ電位判定部５０３において第１の微粒子に対して判定した第１のゼータ電位ζ１と、粒子径判定部５０４において第１の微粒子に対して判定した第１の粒子径ｄ１とを、相関部５０５において第１の微粒子に関するデータとして相互に結び付ける。この相関部５０５が関連付けした結果である粒子相関リストＬＳ２の一例を、図１３に示す。 The correlation unit 505 associates the zeta potential ζ of the fine particles determined by the zeta potential determination unit 503 with the particle size d of the fine particles determined by the particle size determination unit 504. Specifically, the first zeta potential ζ1 determined for the first fine particles in the zeta potential determination unit 503, and the first particle diameter d1 determined for the first fine particles in the particle size determination unit 504 Are correlated with each other as data on the first fine particles in the correlation unit 505. FIG. 13 shows an example of the particle correlation list LS2 that is a result associated with the correlation unit 505.
　図１３は、記憶部５２０が記憶する粒子相関リストＬＳ２の一例を示す図である。この粒子相関リストＬＳ２において、識別部５０２によって付与された粒子番号毎に、粒子径ｄと、ゼータ電位ζとが関連付けられている。相関部５０５は、微粒子Ｐ１について、微粒子Ｐ１の粒子径ｄ１と、微粒子Ｐ１のゼータ電位ζ１とを関連付けて、粒子相関情報ＰＣ１（ｄ１、ζ１）として、粒子相関リストＬＳ２に記憶させる。また、相関部５０５は、微粒子Ｐ２について、微粒子Ｐ２の粒子径ｄ２と、微粒子Ｐ２のゼータ電位ζ２とを関連付けて、粒子相関情報ＰＣ２（ｄ２、ζ２）として、粒子相関リストＬＳ２に記憶させる。
　このように、媒質中に存在する微粒子の状態の相関を判定することができる。 FIG. 13 is a diagram illustrating an example of the particle correlation list LS2 stored in the storage unit 520. In the particle correlation list LS2, the particle diameter d and the zeta potential ζ are associated with each particle number assigned by the identification unit 502. The correlation unit 505 associates the particle diameter d1 of the fine particle P1 with the zeta potential ζ1 of the fine particle P1 and stores it as particle correlation information PC1 (d1, ζ1) in the particle correlation list LS2 for the fine particle P1. Further, the correlation unit 505 associates the particle diameter d2 of the fine particle P2 with the zeta potential ζ2 of the fine particle P2 and stores the particle P2 in the particle correlation list LS2 as particle correlation information PC2 (d2, ζ2).
In this way, the correlation of the state of the fine particles existing in the medium can be determined.
　状態判定部５０６は、相関部５０５が生成した粒子相関リストＬＳ２に基づいて、微粒子の状態を判定する。記憶部５２０には、粒子径ｄの基準範囲と、ゼータ電位ζの基準範囲とをそれぞれ示す基準範囲情報が記憶されている。ここでは、状態判定部５０６による状態判定の一例として、エクソソーム以外の粒子が含まれる試料において、識別部５０２が識別した微粒子を、エクソソームであるか否かを判定する場合について説明する。
　エクソソームの特徴として、粒径が直径３０～２００ｎｍ程度の微粒子であること、そしてまた、構成因子としてシャペロン分子であるＨｓｃ７０、Ｈｓｃ９０やテトラスパニン（ＣＤ９，　ＣＤ６３，　ＣＤ８１）が特異的に存在していることが挙げられる。
　この場合、記憶部５２０には、粒子径のしきい値Ｔｈｄが、基準範囲情報として記憶されている。また、記憶部５２０には、ゼータ電位のしきい値Ｔｈζが、基準範囲情報として記憶されている。これらの場合、記憶部５２０を基準記憶部と言い換えてもよい。このしきい値Ｔｈｄ、及びしきい値Ｔｈζの一例を図１４に示す。 The state determination unit 506 determines the state of the fine particles based on the particle correlation list LS2 generated by the correlation unit 505. The storage unit 520 stores reference range information indicating the reference range of the particle diameter d and the reference range of the zeta potential ζ. Here, as an example of state determination by the state determination unit 506, a case will be described in which it is determined whether or not the microparticles identified by the identification unit 502 are exosomes in a sample containing particles other than exosomes.
The characteristics of exosomes are microparticles with a particle size of about 30 to 200 nm, and the presence of chaperone molecules Hsc70, Hsc90 and tetraspanins (CD9, CD63, CD81) as constituent factors. Is mentioned.
In this case, the storage unit 520 stores a threshold value Thd of particle diameter as reference range information. The storage unit 520 stores a threshold value Thζ of zeta potential as reference range information. In these cases, the storage unit 520 may be rephrased as a reference storage unit. An example of the threshold value Thd and the threshold value Thζ is shown in FIG.
　図１４は、本実施形態の記憶部５２０が記憶するしきい値の一例を示す図である。ここで、一例として、エクソソームの粒子径が、直径３０～１００ｎｍ程度であり、判定対象の微粒子のうち、エクソソーム以外の微粒子の粒子径が、直径１００ｎｍを超える場合について説明する。また、ここでは、一例として、エクソソームのゼータ電位ζが、しきい値Ｔｈζ以下であり、エクソソーム以外の微粒子のゼータ電位ζが、しきい値Ｔｈζを超える場合について説明する。この一例の場合には、状態判定部５０６は、微粒子の粒子径、及び微粒子のゼータ電位ζに基づいて、微粒子の判定を行うことができる。なお、状態判定部５０６が行う微粒子の判定を、微粒子の特定と言い換えてもよい。
　具体的には、この一例の場合、記憶部５２０には、粒子径のしきい値Ｔｈｄとして、１００ｎｍが記憶されている。また、記憶部５２０には、ゼータ電位のしきい値Ｔｈζとして、－６ｍＶが記憶されている。状態判定部５０６は、粒子相関リストＬＳ２に記憶されている粒子相関情報ＰＣのうち、粒子径ｄが、しきい値Ｔｈｄ以下である微粒子であり、かつ、ゼータ電位ζがしきい値Ｔｈζ以下である微粒子を、エクソソームであると判定する。一方、状態判定部５０６は、粒子相関リストＬＳ２に記憶されている粒子相関情報ＰＣのうち、粒子径ｄが、しきい値Ｔｈｄを超える微粒子や、ゼータ電位ζが、しきい値Ｔｈζを超える微粒子を、エクソソームでないと判定する。 FIG. 14 is a diagram illustrating an example of threshold values stored in the storage unit 520 of the present embodiment. Here, as an example, a case will be described in which the particle diameter of exosome is about 30 to 100 nm in diameter, and among the particles to be determined, the particle diameter of particles other than exosome exceeds 100 nm in diameter. Here, as an example, a case will be described in which the zeta potential ζ of the exosome is equal to or lower than the threshold Thζ and the zeta potential ζ of fine particles other than the exosome exceeds the threshold Thζ. In this example, the state determination unit 506 can determine the particle based on the particle diameter of the particle and the zeta potential ζ of the particle. Note that the determination of fine particles performed by the state determination unit 506 may be paraphrased as identification of fine particles.
Specifically, in this example, the storage unit 520 stores 100 nm as the particle size threshold Thd. The storage unit 520 stores −6 mV as the threshold value Thζ of the zeta potential. The state determination unit 506 is a particle whose particle diameter d is equal to or less than the threshold Thd in the particle correlation information PC stored in the particle correlation list LS2, and the zeta potential ζ is equal to or less than the threshold Thζ. A microparticle is determined to be an exosome. On the other hand, the state determination unit 506, among the particle correlation information PC stored in the particle correlation list LS2, fine particles having a particle diameter d exceeding the threshold value Thd, or fine particles having a zeta potential ζ exceeding the threshold value Thζ. Is determined not to be an exosome.
　また、一例として、エクソソームの粒子径が、直径３０～１００ｎｍ程度であり、判定対象の微粒子のうち、エクソソーム以外の微粒子の粒子径が、直径２００ｎｍを超える場合がある。この場合には、一例として、粒子径ｄのしきい値Ｔｈｄを１５０ｎｍにすることにより、状態判定部５０６は、粒子径のみに基づいて微粒子の状態を判定することができる。 Also, as an example, the particle diameter of exosome is about 30 to 100 nm, and among the fine particles to be determined, the particle diameter of fine particles other than exosome may exceed 200 nm. In this case, as an example, by setting the threshold value Thd of the particle diameter d to 150 nm, the state determination unit 506 can determine the state of the fine particles based only on the particle diameter.
　また、直径１００～２００ｎｍの範囲には、エクソソーム以外の微粒子が含まれる場合がある。この場合には、粒子径のしきい値Ｔｈｄ（２００ｎｍ）は、微粒子がエクソソームであるか否かを判定するための一要素として、利用することができる。
　また、直径が２００ｎｍよりも大きい範囲には、単一のエクソソームが含まれていない場合がある。この場合には、粒子径のしきい値Ｔｈｄ（２００ｎｍ）は、微粒子が単一のエクソソームであるか否かを判定するための一要素として、利用することができる。
　また、直径が２００ｎｍよりも大きい範囲には、単一のエクソソームが複数個凝集した微粒子が含まれている場合がある。この場合には、粒子径のしきい値Ｔｈｄ（２００ｎｍ）は、微粒子が単一のエクソソームであるか、凝集したエクソソームであるかを判定するための一要素として、利用することができる。
　このように、基準記憶部において記憶される基準値となるしきい値を、微粒子の状態の判定のための要因として用いることができる。 In addition, fine particles other than exosomes may be included in the diameter range of 100 to 200 nm. In this case, the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether or not the fine particle is an exosome.
Moreover, a single exosome may not be contained in the range whose diameter is larger than 200 nm. In this case, the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether or not the microparticle is a single exosome.
In addition, a range in which the diameter is larger than 200 nm may include fine particles in which a plurality of single exosomes are aggregated. In this case, the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether the microparticle is a single exosome or an aggregated exosome.
As described above, the threshold value serving as the reference value stored in the reference storage unit can be used as a factor for determining the state of the fine particles.
　また、状態判定部５０６は、識別部５０２が識別した微粒子がエクソソームである場合に、このエクソソームが、抗体と反応しているか否かを判定する。この一例の場合、記憶部５２０には、ゼータ電位のしきい値Ｔｈζが、基準範囲情報として記憶されている。上述したように、抗体－エクソソーム複合体のゼータ電位は、エクソソーム単独のゼータ電位と比較して正にシフトしている。この場合、記憶部５２０には、エクソソーム単独のゼータ電位と、抗体－エクソソーム複合体のゼータ電位との間のゼータ電位（例えば、－６ｍｖ）が、ゼータ電位のしきい値Ｔｈζとして記憶されている。状態判定部５０６は、粒子相関リストＬＳ２に記憶されている粒子相関情報ＰＣのうち、微粒子のゼータ電位が、しきい値Ｔｈζ未満である微粒子を、抗体と反応していない単独のエクソソームであると判定する。一方、状態判定部５０６は、粒子相関リストＬＳ２に記憶されている粒子相関情報ＰＣのうち、微粒子のゼータ電位が、しきい値Ｔｈζ以上である微粒子を、抗体－エクソソーム複合体であると判定する。 In addition, the state determination unit 506 determines whether or not the exosome has reacted with the antibody when the microparticles identified by the identification unit 502 are exosomes. In this example, the storage unit 520 stores a threshold value Thζ of zeta potential as reference range information. As described above, the zeta potential of the antibody-exosome complex is positively shifted compared to the zeta potential of exosome alone. In this case, the storage unit 520 stores a zeta potential (eg, −6 mv) between the zeta potential of the exosome alone and the zeta potential of the antibody-exosome complex as the zeta potential threshold Thζ. . The state determination unit 506 determines that, among the particle correlation information PC stored in the particle correlation list LS2, the fine particles whose zeta potential of the fine particles is less than the threshold Thζ is a single exosome that has not reacted with the antibody. judge. On the other hand, in the particle correlation information PC stored in the particle correlation list LS2, the state determination unit 506 determines that the microparticles whose microparticle zeta potential is greater than or equal to the threshold Thζ are antibody-exosome complexes. .
　また、ゼータ電位のしきい値Ｔｈζ（例えば－６ｍＶ）の付近には、単体のエクソソーム及び、抗体－エクソソーム複合体以外の微粒子が存在している場合がある。この場合には、ゼータ電位のしきい値Ｔｈζ（－６ｍＶ）は、微粒子が単体のエクソソームであるか否かを判定するための一要素として、利用することができる。 In addition, there may be a single exosome and fine particles other than the antibody-exosome complex in the vicinity of the threshold value Thζ (for example, −6 mV) of the zeta potential. In this case, the threshold value Thζ (−6 mV) of the zeta potential can be used as one element for determining whether the microparticle is a single exosome.
　また、状態判定部５０６は、粒子径ｄのしきい値とゼータ電位ζのしきい値とを組み合わせて、微粒子の状態を判定することもできる。具体的には、抗体－エクソソーム複合体は、単独のエクソソームと比較して、ゼータ電位が低い。このため、微粒子間に働くクーロン力は、抗体－エクソソーム複合体の方が、単独のエクソソームと比較して、弱い。この微粒子間に働くクーロン力は、微粒子間の間隔を遠ざける斥力として作用する。つまり、抗体－エクソソーム複合体の方が、単独のエクソソームと比較して、微粒子間に働く斥力が弱い。このため、抗体－エクソソーム複合体の方が、単独のエクソソームと比較して、凝集しやすい傾向がある。ここで、微粒子どうしが凝集すると、凝集した複数の微粒子が１つの微粒子として振る舞うため、ブラウン運動の運動量に変化が生じる。したがって、粒子径判定部５０４は、凝集した複数の微粒子を１つの微粒子として判定することにより、凝集していない場合に比べて、粒子径ｄが大きくなる方向にシフトする。
　ここで、粒子径ｄが２００ｎｍ以下の微粒子をエクソソームであると判定する場合を一例にして説明する。粒子径判定部５０４は、抗体－エクソソーム複合体を直径が２００ｎｍを超える微粒子であると判定することがある。このため、状態判定部５０６が粒子径ｄのみによって判定した場合には、抗体－エクソソーム複合体の粒子径ｄが、エクソソームであるか否かの粒子径ｄのしきい値Ｔｈｄを超えるため、抗体－エクソソーム複合体がエクソソームではないと判定される場合がある。そこで、状態判定部５０６は、粒子径ｄが２００ｎｍ以下の微粒子をエクソソームであると判定するとともに、粒子径ｄが２００ｎｍを超える微粒子であっても、ゼータ電位ζがしきい値Ｔｈζ以下である場合には、その微粒子をエクソソームであると判定する。つまり、状態判定部５０６は、粒子径ｄのしきい値Ｔｈｄとゼータ電位ζのしきい値Ｔｈζとを組み合わせて、微粒子がエクソソームであるか否かを判定する。 The state determination unit 506 can also determine the state of the fine particles by combining the threshold value of the particle diameter d and the threshold value of the zeta potential ζ. Specifically, the antibody-exosome complex has a low zeta potential compared to a single exosome. For this reason, the Coulomb force acting between the microparticles is weaker in the antibody-exosome complex than in the single exosome. The Coulomb force acting between the fine particles acts as a repulsive force that keeps the fine particles apart. That is, the antibody-exosome complex has less repulsive force acting between the microparticles than the single exosome. For this reason, the antibody-exosome complex tends to aggregate more easily than a single exosome. Here, when the fine particles are aggregated, a plurality of the aggregated fine particles behave as one fine particle, so that the momentum of the Brownian motion is changed. Therefore, the particle diameter determination unit 504 shifts the particle diameter d to be larger than that in the case where the particles are not aggregated by determining a plurality of aggregated fine particles as one fine particle.
Here, a case where a fine particle having a particle diameter d of 200 nm or less is determined to be an exosome will be described as an example. The particle size determination unit 504 may determine that the antibody-exosome complex is a fine particle having a diameter exceeding 200 nm. Therefore, when the state determination unit 506 determines only by the particle diameter d, the particle diameter d of the antibody-exosome complex exceeds the threshold value Thd of the particle diameter d whether or not the antibody is an exosome. -It may be determined that the exosome complex is not an exosome. Therefore, the state determination unit 506 determines that a fine particle having a particle diameter d of 200 nm or less is an exosome, and the zeta potential ζ is equal to or less than a threshold Thζ even if the particle diameter d is greater than 200 nm. Is determined to be an exosome. That is, the state determination unit 506 determines whether or not the fine particle is an exosome by combining the threshold value Thd of the particle diameter d and the threshold value Thζ of the zeta potential ζ.
　また、微粒子がエクソソームであるか否かの判定には、テトラスパニン（ＣＤ９，ＣＤ８１など）のように、エクソソームに特異的に結合される抗体を利用することができる。
　つまり、エクソソームに対して抗体を作用させることによるゼータ電位ζ及び粒子径ｄの、それぞれの変化に基づいて、微粒子がエクソソームであるか否かを判定することができる。
　粒子検出システム１は、ゼータ電位ζ及び粒子径ｄに基づく、上述のような評価条件を様々に組み合わせて、微粒子の評価を行うことができる利点を有する。 For determining whether or not the microparticle is an exosome, an antibody that specifically binds to the exosome, such as tetraspanin (CD9, CD81, etc.) can be used.
That is, it is possible to determine whether or not the microparticle is an exosome based on the respective changes in the zeta potential ζ and the particle diameter d caused by causing the antibody to act on the exosome.
The particle detection system 1 has an advantage that fine particles can be evaluated by variously combining the above-described evaluation conditions based on the zeta potential ζ and the particle diameter d.
　状態判定部５０６は、粒子径ｄのしきい値Ｔｈｄとゼータ電位ζのしきい値Ｔｈζとを組み合わせた上で、識別部５０２によるトラッキングの結果に基づいて、微粒子の状態を判定することもできる。具体的には、状態判定部５０６は、単独のエクソソームに抗体を反応させ、さらに抗体－エクソソーム複合体どうしが凝集するまでの経過を、識別部５０２によるトラッキングの結果に基づいて追跡する。具体的には、状態判定部５０６は、各微粒子の粒子径ｄとゼータ電位ζとが、時間の経過により、図１４に示す領域ＤＭ１～領域ＤＭ４のうち、いずれの領域からいずれの領域に移動するかによって、微粒子の状態を判定する。一例として、状態判定部５０６は、領域ＤＭ３に存在する微粒子（例えば、単独のエクソソーム）が、領域ＤＭ２に移動した場合には、エクソソームが抗体と反応して、抗体－エクソソーム複合体に変化したと判定する。また、状態判定部５０６は、このエクソソームが、領域ＤＭ２から領域ＤＭ１に移動した場合には、抗体－エクソソーム複合体どうしが凝集したと判定する。 The state determination unit 506 can determine the state of the fine particles based on the result of tracking by the identification unit 502 after combining the threshold value Thd of the particle diameter d and the threshold value Thζ of the zeta potential ζ. . Specifically, the state determination unit 506 tracks the progress until the antibody reacts with a single exosome and further aggregates the antibody-exosome complexes based on the result of tracking by the identification unit 502. Specifically, the state determination unit 506 moves the particle diameter d and the zeta potential ζ of each fine particle from any of the regions DM1 to DM4 shown in FIG. The state of the fine particles is determined depending on whether or not As an example, the state determination unit 506 determines that when a microparticle (for example, a single exosome) present in the region DM3 moves to the region DM2, the exosome reacts with the antibody and changes to an antibody-exosome complex. judge. Further, when the exosome moves from the region DM2 to the region DM1, the state determination unit 506 determines that the antibody-exosome complexes are aggregated.
　評価部５０７は、微粒子の状態の良否を評価する。一例として、評価部５０７は、状態判定部５０６が判定した微粒子の状態に基づいて、微粒子の状態をＡランク、Ｂランク、Ｃランクにランク付けする。ここで、Ａランクとは、微粒子の粒子径ｄ及びゼータ電位ζのいずれもが、基準範囲内に含まれる場合である。また、Ｂランクとは、微粒子の粒子径ｄ及びゼータ電位ζのいずれか一方が基準範囲内に含まれない場合である。また、Ｃランクとは、微粒子の粒子径ｄ及びゼータ電位ζのいずれもが基準範囲内に含まれない場合である。 Evaluation unit 507 evaluates the quality of the fine particles. As an example, the evaluation unit 507 ranks the state of the fine particles into A rank, B rank, and C rank based on the state of the fine particles determined by the state determination unit 506. Here, the A rank is a case where both the particle diameter d and the zeta potential ζ of the fine particles are included in the reference range. The rank B is a case where either one of the particle diameter d and the zeta potential ζ of the fine particles is not included in the reference range. The C rank is a case where neither the particle diameter d of the fine particles nor the zeta potential ζ is included in the reference range.
　一例として、評価部５０７が、微粒子が単独のエクソソームであるか否かを評価する場合について説明する。この場合、評価部５０７は、微粒子が領域ＤＭ３に存在する場合には、この微粒子のランクを、ランクＡであると判定する。また、評価部５０７は、微粒子が領域ＤＭ２又は領域ＤＭ４に存在する場合には、この微粒子のランクを、ランクＢであると判定する。また、評価部５０７は、微粒子が領域ＤＭ１に存在する場合には、この微粒子のランクを、ランクＣであると判定する。 As an example, a case where the evaluation unit 507 evaluates whether or not the microparticle is a single exosome will be described. In this case, the evaluation unit 507 determines that the rank of the fine particles is rank A when the fine particles are present in the region DM3. Further, the evaluation unit 507 determines that the rank of the fine particles is rank B when the fine particles are present in the region DM2 or the region DM4. The evaluation unit 507 determines that the rank of the fine particles is rank C when the fine particles are present in the region DM1.
［制御装置の動作］
　次に、図１５を参照して、制御装置５の動作について説明する。
　図１５は、制御装置５の動作の一例を示す図である。ここでは、粒子検出装置１００が、所定の時間間隔によって側方散乱光の画像を撮像する場合について説明する。 [Operation of control device]
Next, the operation of the control device 5 will be described with reference to FIG.
FIG. 15 is a diagram illustrating an example of the operation of the control device 5. Here, a case where the particle detection apparatus 100 captures an image of side scattered light at a predetermined time interval will be described.
　取得部５０１は、粒子検出装置１００の撮像部３２が撮像した画像を、粒子検出装置１００から１枚ずつ取得する（ステップＳ１０）。この画像には、泳動流路１５０を電気泳動する微粒子の画像が含まれている。また、この微粒子の画像には、エクソソームの画像が含まれている。 The acquisition unit 501 acquires images captured by the imaging unit 32 of the particle detection device 100 one by one from the particle detection device 100 (step S10). This image includes an image of fine particles that are electrophoresed in the migration channel 150. The fine particle image includes an exosome image.
　次に、識別部５０２は、ステップＳ１０において取得された画像の中から、微粒子の画像を抽出し、微粒子毎に固有の粒子番号を付与する。つまり、識別部５０２は、微粒子をラベリングする（ステップＳ２０）。識別部５０２は、すべての撮像済み画像について、ラベリングが終了したか否かを判定する（ステップＳ３０）。識別部５０２は、すべての撮像済み画像について、ラベリングが終了していないと判定した場合（ステップＳ３０；ＮＯ）には、処理をステップＳ１０に戻し、次の画像についてラベリングを行う。識別部５０２は、すべての撮像済み画像について、ラベリングが終了したと判定した場合（ステップＳ３０；ＹＥＳ）には、処理をステップＳ４０に進め、識別した微粒子についてトラッキングを行う。 Next, the identification unit 502 extracts a fine particle image from the image acquired in step S10, and assigns a unique particle number to each fine particle. That is, the identification unit 502 labels the fine particles (step S20). The identification unit 502 determines whether or not labeling has been completed for all captured images (step S30). If it is determined that the labeling has not been completed for all captured images (step S30; NO), the identification unit 502 returns the process to step S10 and performs the labeling for the next image. If it is determined that the labeling has been completed for all captured images (step S30; YES), the identification unit 502 advances the process to step S40 and performs tracking for the identified fine particles.
　次に、ゼータ電位判定部５０３は、識別部５０２がトラッキングした結果に基づいて、微粒子毎のゼータ電位ζを判定する（ステップＳ５０）。また、粒子径判定部５０４は、識別部５０２がトラッキングした結果に基づいて、微粒子毎の粒子径を判定する（ステップＳ６０）。なお、ステップＳ５０と、ステップＳ６０とは、順序が逆であってもよく、並列して実行されてもよい。 Next, the zeta potential determination unit 503 determines the zeta potential ζ for each fine particle based on the result tracked by the identification unit 502 (step S50). Further, the particle size determination unit 504 determines the particle size for each fine particle based on the result of tracking by the identification unit 502 (step S60). Note that the order of step S50 and step S60 may be reversed or may be executed in parallel.
　次に、相関部５０５は、ゼータ電位判定部５０３が判定した微粒子のゼータ電位ζと、粒子径判定部５０４が判定した微粒子の粒子径ｄとを関連付ける（ステップＳＳ７０）。
　相関部５０５は、関連付けした結果を示す粒子相関リストＬＳ２を生成し、生成した粒子相関リストＬＳ２を記憶部５２０に記憶させる。相関部５０５は、すべての微粒子について関連付けが終了していないと判定した場合（ステップＳ８０；ＮＯ）には、処理をステップＳ４０に戻す。相関部５０５は、すべての微粒子について関連付けが終了したと判定した場合(ステップＳ８０；ＹＥＳ）には、処理をステップＳ９０に進める。 Next, correlation section 505 associates the zeta potential ζ of the fine particles determined by zeta potential determination section 503 with the particle diameter d of the fine particles determined by particle diameter determination section 504 (step SS70).
The correlation unit 505 generates a particle correlation list LS2 indicating the associated result, and stores the generated particle correlation list LS2 in the storage unit 520. When the correlation unit 505 determines that the association has not been completed for all the fine particles (step S80; NO), the correlation unit 505 returns the process to step S40. When the correlation unit 505 determines that the association has been completed for all the fine particles (step S80; YES), the correlation unit 505 advances the process to step S90.
　次に、状態判定部５０６及び評価部５０７は、ステップＳ７０において生成された粒子相関リストＬＳ２に基づいて、粒子の状態の判定及び評価を行う。 Next, the state determination unit 506 and the evaluation unit 507 perform particle state determination and evaluation based on the particle correlation list LS2 generated in step S70.
　上記の粒子検出装置１００は、細胞外小胞のゼータ電位を測定するための装置の一例であり、細胞外小胞の集団中の個々の細胞外小胞のゼータ電位を測定可能な装置であれば、特に制限なく利用することができる。上記のような構成の粒子検出装置、又は他のゼータ電位測定装置を用いて、細胞外小胞の集団中の個々の細胞外小胞のゼータ電位を測定し、それらのゼータ電位の標準偏差を算出して、細胞外小胞の集団のゼータ電位の標準偏差とすることができる。前記のように算出された細胞外小胞のゼータ電位の標準偏差が５ｍＶ以下である場合には、本実施形態の細胞外小胞の集団であると判定される。 The particle detection apparatus 100 is an example of an apparatus for measuring the zeta potential of extracellular vesicles, and can be a device capable of measuring the zeta potential of individual extracellular vesicles in a population of extracellular vesicles. Can be used without any particular restrictions. Using the particle detector configured as described above, or other zeta potential measuring device, the zeta potential of individual extracellular vesicles in a population of extracellular vesicles is measured, and the standard deviation of those zeta potentials is calculated. It can be calculated as the standard deviation of the zeta potential of the population of extracellular vesicles. When the standard deviation of the zeta potential of the extracellular vesicle calculated as described above is 5 mV or less, it is determined that the group is the extracellular vesicle group of the present embodiment.
　本実施形態の細胞外小胞の集団は、ゼータ電位の標準偏差が５ｍＶ以下であり、細胞外小胞の状態、特に細胞外小胞の表面状態に関して均質性の高い細胞外小胞の集団である。そのため、医薬品、化粧品、食品等の様々な用途に利用することができる。本実施形態の細胞外小胞の集団は、当該集団を構成する細胞外小胞の品質が揃っているため、特に均質性の高さが求められる医薬品としても、好適に利用することができる。医薬品用途としては、例えば、薬物を内包するキャリアとしての利用や、間葉系幹細胞等の特定の細胞由来のエクソソームの薬物としての利用等が挙げられるが、これらに限定されない。
　ゼータ電位の測定を、細胞外小胞に特異的結合物質を結合させた状態で行った場合には、特に当該特異的結合物質の結合対象である分子の発現状態に関して均質性の高い細胞外小胞の集団を得ることができる。例えば、細胞外小胞の膜表面を、疾患部位などを標的とする分子（例えば、がん細胞膜表面抗原に対する抗体）で修飾した場合には、当該分子に対する特異的結合物質を用いてゼータ電位の測定を行い、当該ゼータ電位の標準偏差が５ｍＶ以下である細胞外小胞の集団を得てもよい。そのような細胞外小胞の集団を構成する細胞外小胞は、当該分子による膜表面の修飾状態に関して特に均質性が高い。そのため、当該疾患部位へのＤＤＳのキャリアとして好適に用いることができる。 The population of extracellular vesicles of this embodiment is a population of extracellular vesicles having a standard deviation of zeta potential of 5 mV or less and high homogeneity with respect to the state of extracellular vesicles, particularly the surface state of extracellular vesicles. is there. Therefore, it can be used for various uses such as pharmaceuticals, cosmetics, and foods. The extracellular vesicle population of this embodiment can be suitably used as a pharmaceutical product that requires particularly high homogeneity because the quality of the extracellular vesicles constituting the population is uniform. Examples of the pharmaceutical use include, but are not limited to, use as a carrier encapsulating a drug and use of a specific cell-derived exosome such as a mesenchymal stem cell as a drug.
When the zeta potential is measured in a state in which a specific binding substance is bound to an extracellular vesicle, the extracellular smallness with high homogeneity particularly with respect to the expression state of the molecule to which the specific binding substance is bound. A population of cells can be obtained. For example, when the membrane surface of an extracellular vesicle is modified with a molecule that targets a disease site or the like (for example, an antibody against a cancer cell membrane surface antigen), the zeta potential can be reduced using a specific binding substance for the molecule. Measurement may be performed to obtain a population of extracellular vesicles having a standard deviation of the zeta potential of 5 mV or less. Extracellular vesicles constituting such a population of extracellular vesicles are particularly homogeneous with respect to the state of modification of the membrane surface by the molecule. Therefore, it can be suitably used as a carrier for DDS to the disease site.
＜細胞外小胞の集団の製造方法＞
　１実施形態において、本発明は、細胞外小胞の集団の製造方法を提供する。本実施形態の方法は、（ａ）複数の細胞の細胞周期を同調させる工程と、（ｂ）前記工程（ａ）後、前記複数の細胞の培地を、細胞外小胞を実質的に含まない培地に交換する工程と、（ｃ）前記培地交換した培地で、前記複数の細胞を培養する工程と、（ｄ）前記工程（ｃ）後の培地から、細胞外小胞の集団を回収する工程と、を含む。 <Method for producing extracellular vesicle population>
In one embodiment, the present invention provides a method for producing a population of extracellular vesicles. The method of the present embodiment includes (a) a step of synchronizing the cell cycle of a plurality of cells, and (b) a medium of the plurality of cells substantially not including extracellular vesicles after the step (a). Replacing the medium; (c) culturing the plurality of cells in the medium replaced; and (d) recovering a population of extracellular vesicles from the medium after the step (c). And including.
［工程（ａ）］
　工程（ａ）は、複数の細胞の細胞周期を同調させる工程である。
　前記複数の細胞は、全て同一種類の細胞であることが好ましい。同一種類の細胞を培養することにより、品質が揃った細胞外小胞を得ることができる。細胞の種類は、細胞外小胞を放出する限り、特に限定されない。細胞としては、例えば、腫瘍細胞などの各種疾患細胞；樹状細胞、Ｔ細胞、Ｂ細胞などの免疫細胞；神経細胞などの各種組織細胞；脂肪細胞；間葉系幹細胞、造血幹細胞などの体性幹細胞；ＥＳ細胞、ｉＰＳ細胞などの多能性幹細胞；生殖細胞等が挙げられるが、これらに限定されない。前記細胞が由来する生物種も、特に限定されない。細胞が由来する生物種も特に限定されず、ヒト、及びヒト以外の哺乳類（例えば、マウス、モルモット、サル、イヌ、ネコ、ウシ、ウマ、ブタ等）の細胞等が挙げられる。細胞は、製造するエクソソームの用途に応じて適宜選択すればよく、例えば、再生医療に利用する場合には、ヒトの間葉系幹細胞等を選択することができる。
　「複数の細胞」は、２個以上の細胞であれば特に限定されず、例えば、１０２個以上、１０３個以上、１０４個以上等の細胞であってもよい。複数の細胞は、例えば、１０２～１０１５個、１０３～１０１２個、又は１０４～１０１０個等の細胞であってもよい。 [Step (a)]
Step (a) is a step of synchronizing the cell cycle of a plurality of cells.
The plurality of cells are preferably the same type of cells. By culturing the same type of cells, extracellular vesicles of uniform quality can be obtained. The cell type is not particularly limited as long as it releases extracellular vesicles. Examples of cells include various disease cells such as tumor cells; immune cells such as dendritic cells, T cells and B cells; various tissue cells such as nerve cells; fat cells; mesenchymal stem cells and hematopoietic stem cells. Examples include, but are not limited to, stem cells; pluripotent stem cells such as ES cells and iPS cells; germ cells and the like. The biological species from which the cells are derived is not particularly limited. The biological species from which the cells are derived is not particularly limited, and examples thereof include cells of humans and mammals other than humans (for example, mice, guinea pigs, monkeys, dogs, cats, cows, horses, pigs, etc.). The cells may be appropriately selected according to the use of the exosome to be produced. For example, when used for regenerative medicine, human mesenchymal stem cells and the like can be selected.
"More cells", if two or more cells is not particularly limited, for example, 10 2 or more, 10 3 or more, may be a cell, such as a 10 4 or more. The plurality of cells may be, for example, 10 2 to 10 15 cells, 10 3 to 10 12 cells, or 10 4 to 10 10 cells.
　細胞周期は、間期とＭ期とに分けられ、間期はさらにＧ１期、Ｓ期、Ｇ２期とに分けられる（図１６参照）。本工程において、複数の細胞を同調させる細胞周期は、特に限定されず、Ｇ１期、Ｓ期、Ｇ２期、及びＭ期のいずれであってもよい。また、Ｇ１期／Ｓ期の境界等、２つの周期の境界に同調させてもよい。 The cell cycle is divided into an interphase and an M phase, and the interphase is further divided into a G1 phase, an S phase, and a G2 phase (see FIG. 16). In this step, the cell cycle for synchronizing a plurality of cells is not particularly limited, and may be any of G1 phase, S phase, G2 phase, and M phase. Further, it may be synchronized with the boundary of two periods such as the boundary of G1 period / S period.
　前記複数の細胞の細胞周期を同調させる方法は、特に限定されず、公知の方法を用いることができる。そのような方法としては、例えば、細胞周期同調剤を含む培地で培養する方法、コンフルエントな状態で細胞を培養する方法、血清飢餓状態で細胞を培養する方法、チミジンブロック法等が挙げられる。 The method for synchronizing the cell cycle of the plurality of cells is not particularly limited, and a known method can be used. Examples of such a method include a method of culturing in a medium containing a cell cycle synchronizer, a method of culturing cells in a confluent state, a method of culturing cells in a serum-starved state, and a thymidine block method.
（細胞周期同調剤を用いる方法）
　「細胞周期同調剤」とは、複数の細胞の細胞周期を同調させる作用を有する薬剤である。細胞周期同調剤としては、特定の細胞周期で細胞周期の進行を停止させる作用を有する薬剤等が挙げられる。細胞周期同調剤は、公知のものを特に制限なく用いることができる。一例として、細胞をＧ１期に同調させる細胞周期同調剤として、レプトマイシンＡ及びレプトマイシンＢ等が挙げられる。例えば、細胞周期同調剤を培地に添加し、複数の細胞を培養する。培養培地は、細胞の種類に応じて適宜選択すればよい。例えば、ヒトの細胞であれば、公知のヒト細胞培養用培地を特に制限なく用いることができる。ヒト細胞培養用培地としては、一例として、ウシ胎児血清（ＦＢＳ）等を添加したＲＰＭＩ培地等が挙げられる。 (Method using cell cycle synchronizer)
The “cell cycle synchronizer” is an agent having an action of synchronizing the cell cycle of a plurality of cells. Examples of the cell cycle synchronizer include a drug having an action of stopping the progression of the cell cycle in a specific cell cycle. A well-known thing can be especially used for a cell cycle synchronizing agent without a restriction | limiting. As an example, leptomycin A, leptomycin B, etc. are mentioned as a cell cycle synchronizer which synchronizes a cell to G1 phase. For example, a cell cycle synchronizer is added to a culture medium, and a plurality of cells are cultured. What is necessary is just to select a culture medium suitably according to the kind of cell. For example, in the case of human cells, a known human cell culture medium can be used without any particular limitation. An example of the human cell culture medium is RPMI medium to which fetal bovine serum (FBS) or the like is added.
　細胞周期同調剤を含む培地での培養時間は、細胞の種類に応じて適宜選択すればよい。好ましくは、細胞周期が１周期進行する時間以上、培養することが好ましい。培養時間としては、例えば、１０時間以上、１５時間以上、又は２０時間以上等が例示される。培養時間の上限は、特に限定されないが、細胞周期が同調した後に長時間培養する必要はないため、例えば、細胞周期が５周期、４周期、３周期、又は２周期進行するよりも短い時間等が挙げられる。培養時間の上限の具体例としては、例えば、５０時間以内、４０時間以内、又は３０時間以内等が例示される。一例として、培養時間は、２４時間とすることができる。 The culture time in a medium containing a cell cycle synchronizer may be appropriately selected according to the cell type. It is preferable to culture for a period of time longer than the cell cycle proceeds for one cycle. Examples of the culture time include 10 hours or more, 15 hours or more, or 20 hours or more. The upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 5 cycles, 4 cycles, 3 cycles, or 2 cycles, etc. Is mentioned. Specific examples of the upper limit of the culture time include, for example, within 50 hours, within 40 hours, or within 30 hours. As an example, the culture time can be 24 hours.
　培養開始時の細胞濃度は、特に限定されないが、例えば、例えば、１０３～１０１０個／ｍＬ、１０３～１０９個／ｍＬ、１０４～１０９個／ｍＬ等であってもよい。 The cell concentration at the start of the culture is not particularly limited, and may be, for example, 10 3 to 10 10 cells / mL, 10 3 to 10 9 cells / mL, 10 4 to 10 9 cells / mL, and the like.
　細胞の培養条件は、特に限定されず、細胞の種類に応じて、当該細胞の培養に一般的に用いられる条件を用いればよい。例えば、温度条件としては、２５～４０℃、３０～３７℃等が例示される。 The cell culturing conditions are not particularly limited, and conditions generally used for culturing the cells may be used according to the cell type. For example, the temperature conditions include 25 to 40 ° C., 30 to 37 ° C. and the like.
（コンフルエントな状態で細胞を培養する方法）
　「コンフルエントな状態」とは、培養容器内で増殖可能な濃度に細胞が達し、細胞の増殖がほぼ停止しした状態をいう。コンフルエントな状態で細胞を培養することにより、細胞周期をＧ１期に同調させることができる。 (Method of culturing cells in a confluent state)
The “confluent state” refers to a state in which cells reach a concentration capable of growing in a culture container and cell growth is almost stopped. By culturing cells in a confluent state, the cell cycle can be synchronized with the G1 phase.
　例えば、シャーレ等の培養容器内で細胞を培養すると、細胞が増殖してコンフルエントな状態に達する。例えば、シャーレ等で培養する場合、シャーレ全体に細胞が広がった時点で、コンフルエントな状態に達したと判断することができる。その状態で、培養を継続することにより、細胞周期を同調させることができる。コンフルエントな状態に達してからの培養時間は、上記と同様に、細胞周期が１周期進行する時間以上、培養することが好ましい。培養時間としては、例えば、１０時間以上、１５時間以上、２０時間以上等が例示される。培養時間の上限は、特に限定されないが、細胞周期が同調した後に長時間培養する必要はないため、例えば、細胞周期が４周期、３周期、又は２周期進行するよりも短い時間等が挙げられる。培養時間の上限の具体例としては、例えば、５０時間以内、４０時間以内、３０時間以内とすることができる。一例として、培養時間は、２４時間とすることができる。 For example, when cells are cultured in a culture container such as a petri dish, the cells proliferate and reach a confluent state. For example, when culturing in a petri dish or the like, it can be determined that a confluent state has been reached when cells have spread throughout the petri dish. In this state, the cell cycle can be synchronized by continuing the culture. The culture time after reaching the confluent state is preferably cultivated for at least the time required for one cycle of the cell cycle, as described above. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like. The upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. . Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours. As an example, the culture time can be 24 hours.
　培養の方法は、細胞周期同調剤を含まない培地を用いる以外は、上記（細胞周期同調剤を用いる方法）で記載した方法と同様に行えばよい。 The culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium not containing cell cycle synchronizer is used.
（血清飢餓状態で培養する方法）
　血清飢餓状態での培養は、血清を含まない培地で細胞を培養すればよく、公知の方法により行うことができる。血清を含まない培地は、例えば、細胞の種類に応じて適宜選択される培地の組成において、血清を無添加とすることにより調製することができる。血清飢餓状態で細胞を培養することにより、細胞周期をＧ１期に同調させることができる。
　血清飢餓状態での培養時間は、上記と同様に、細胞周期が１周期進行する時間以上、培養することが好ましい。培養時間としては、例えば、１０時間以上、１５時間以上、２０時間以上等が例示される。培養時間の上限は、特に限定されないが、細胞周期が同調した後に長時間培養する必要はないため、例えば、細胞周期が４周期、３周期、又は２周期進行するよりも短い時間等が挙げられる。培養時間の上限の具体例としては、例えば、５０時間以内、４０時間以内、３０時間以内とすることができる。一例として、培養時間は、２４時間とすることができる。 (Method of culturing in serum-starved state)
The culture in the serum starvation state may be performed by a known method by culturing cells in a medium not containing serum. A medium not containing serum can be prepared, for example, by adding no serum in the composition of the medium appropriately selected according to the cell type. By culturing cells in serum-starved state, the cell cycle can be synchronized with the G1 phase.
The culture time in the serum starvation state is preferably cultivated for a time longer than the time required for one cycle of the cell cycle as described above. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like. The upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. . Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours. As an example, the culture time can be 24 hours.
　培養の方法は、血清を含まない培地を用いる以外は、上記（細胞周期同調剤を用いる方法）で記載した方法と同様に行えばよい。 The culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium not containing serum is used.
（チミジンブロック法）
　チミジンブロックは、チミジンを過剰量含む培地で、細胞を培養すればよく、公知の方法により行うことができる。チミジンブロックを行なうことにより、細胞周期をＳ期に同調させることができる。培地中のチミジンの濃度としては、例えば、１～５ｍＭ、１．５～４ｍＭ、２～３ｍＭ等が例示される。培地は、チミジンを過剰に添加する以外は、細胞の種類に応じて適宜選択される培地を用いればよい。
　チミジンを過剰に含む培地での培養時間は、上記と同様に、細胞周期が１周期進行する時間以上、培養することが好ましい。培養時間としては、例えば、１０時間以上、１５時間以上、２０時間以上等が例示される。培養時間の上限は、特に限定されないが、細胞周期が同調した後に長時間培養する必要はないため、例えば、細胞周期が４周期、３周期、又は２周期進行するよりも短い時間等が挙げられる。培養時間の上限の具体例としては、例えば、５０時間以内、４０時間以内、３０時間以内とすることができる。一例として、培養時間は、２４時間とすることができる。 (Thymidine block method)
The thymidine block may be performed by a known method by culturing cells in a medium containing an excessive amount of thymidine. By performing thymidine blocking, the cell cycle can be synchronized to the S phase. Examples of the concentration of thymidine in the medium include 1 to 5 mM, 1.5 to 4 mM, and 2 to 3 mM. The medium may be appropriately selected according to the cell type, except that thymidine is added in excess.
As in the above, the culture time in the medium containing excess thymidine is preferably cultivated for at least the time required for one cycle of the cell cycle. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like. The upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. . Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours. As an example, the culture time can be 24 hours.
　また、ダブルチミジンブロック法を用いて、細胞周期を同調させてもよい。ダブルチミジンブロック法もまた、公知の方法で行うことができる。ダブルチミジンブロック法では、チミジンブロックを２回行う。例えば、チミジンを過剰に含む培地で一定期間（例えば、１０～３０時間程度）培養した後、培地を交換し、チミジンを含まない培地で一定期間（例えば、６～２０時間程度）培養を行なう。その後、再度、チミジンを過剰に含む培地で培養することにより、細胞周期をＧ１期／Ｓ期の境界に同調させることができる。 Alternatively, the cell cycle may be synchronized using the double thymidine block method. The double thymidine block method can also be performed by a known method. In the double thymidine block method, thymidine block is performed twice. For example, after culturing in a medium containing excess thymidine for a certain period (for example, about 10 to 30 hours), the medium is changed, and culturing is performed for a certain period (for example, about 6 to 20 hours) in a medium not containing thymidine. Thereafter, the cell cycle can be synchronized with the boundary between G1 phase / S phase by culturing again in a medium containing excess thymidine.
　培養の方法は、チミジンを過剰に含む培地を用いる以外は、上記（細胞周期同調剤を用いる方法）で記載した方法と同様に行えばよい。 The culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium containing excess thymidine is used.
［工程（ｂ）］
　工程（ｂ）は、前記工程（ａ）後、前記複数の細胞の培地を、細胞外小胞を実質的に含まない培地に交換する工程である。
　培地の交換は、例えば、工程（ａ）における培養容器から培養上清を除去し、細胞外小胞を実質的に含まない培地を添加することにより行うことができる。また、培地上清を除去後に、細胞外小胞を実質的に含まない培地で１～３回程度細胞の洗浄を行ってもよい。細胞が浮遊細胞等である場合には、遠心分離やフィルターろ過等によって細胞を回収し、適宜細胞の洗浄を行って、細胞外小胞を実質的に含まない培地に細胞を播種してもよい。 [Step (b)]
Step (b) is a step of replacing the medium of the plurality of cells with a medium substantially free of extracellular vesicles after the step (a).
The medium can be exchanged, for example, by removing the culture supernatant from the culture vessel in step (a) and adding a medium substantially free of extracellular vesicles. Further, after removing the medium supernatant, the cells may be washed about 1 to 3 times with a medium substantially free of extracellular vesicles. When the cells are floating cells or the like, the cells may be collected by centrifugation, filter filtration, etc., washed appropriately, and seeded in a medium substantially free of extracellular vesicles. .
　「細胞外小胞を実質的に含まない培地」とは、培地成分中に細胞外小胞を実質的に含まない培地である。「細胞外小胞を実質的に含まない」とは、細胞外小胞を全く含まないか、細胞外小胞を含んでいたとしても無視できる程度の含有量であることをいう。細胞外小胞を実質的に含まない培地としては、培地中に含まれる細胞外小胞の濃度が、例えば、０～１０個／ｍＬ程度、好ましくは０～５個／ｍＬ程度、より好ましくは０～３個／ｍＬ程度、さらに好ましくは０～１個／ｍＬ程度、特に好ましくは０～０．５個／ｍＬ程度である培地が挙げられる。
　例えば、血清等の生物由来成分を含む培地では、培地中に、生物の細胞由来の細胞外小胞が存在する。そのため、そのような培地で細胞の培養を行なうと、培地中には、当該細胞から放出された細胞外小胞と、元々培地中に含まれる細胞外小胞とが混在することになり、品質の揃った細胞外小胞を得られない。そのため、本工程では、細胞培養培地を、細胞外小胞を実質的に含まない培地に交換する。
　本工程により、細胞周期が同調する前に放出された細胞外小胞を除去できると共に、培地からの細胞外小胞の持ち込みを排除することができる。 The “medium substantially free of extracellular vesicles” is a medium substantially free of extracellular vesicles in the medium components. “Substantially free of extracellular vesicles” means that the content is negligible even if extracellular vesicles are contained, or no extracellular vesicles. As a medium substantially free of extracellular vesicles, the concentration of extracellular vesicles contained in the medium is, for example, about 0 to 10 cells / mL, preferably about 0 to 5 cells / mL, more preferably Examples of the medium include about 0 to 3 / mL, more preferably about 0 to 1 / mL, and particularly preferably about 0 to 0.5 / mL.
For example, in a culture medium containing biological components such as serum, extracellular vesicles derived from biological cells are present in the culture medium. Therefore, when cells are cultured in such a medium, the medium contains both extracellular vesicles released from the cells and extracellular vesicles originally contained in the medium. It is not possible to obtain extracellular vesicles with uniform size. Therefore, in this step, the cell culture medium is replaced with a medium that is substantially free of extracellular vesicles.
By this step, it is possible to remove extracellular vesicles released before the cell cycle is synchronized, and to eliminate the introduction of extracellular vesicles from the medium.
　細胞外小胞を実質的に含まない培地の作製方法は、特に限定されず、公知の細胞外小胞の回収方法を適用することができる。例えば、培地を調製後、超遠心分離、限外ろ過等を行なうことにより、培地中の細胞外小胞を除去することができる。したがって、細胞外小胞を実質的に含まない培地は、培地の調整後、培地中の細胞外小胞を除去する処理を行った培地であるということもできる。 The method for producing a medium substantially free of extracellular vesicles is not particularly limited, and known methods for recovering extracellular vesicles can be applied. For example, after the medium is prepared, extracellular vesicles in the medium can be removed by performing ultracentrifugation, ultrafiltration, or the like. Therefore, it can also be said that the medium substantially free of extracellular vesicles is a medium that has been subjected to a treatment for removing extracellular vesicles in the medium after the medium has been adjusted.
［工程（ｃ）］
　工程（ｃ）は、前記培地交換した培地で、前記複数の細胞を培養する工程である。
　細胞の培養は、細胞外小胞を含まない培地で培養すること以外は、上記工程（ａ）で記載した方法と同様に行うことができる。本工程での培養時間は、細胞が細胞外小胞を放出するのに十分な時間であればよい。例えば、３０分以上、１時間以上、１．５時間以上とすることができる。また、例えば、３０分～５時間、１～４時間、１．５～３時間等であってもよい。例えば、培養時間は、２時間とすることができる。 [Step (c)]
Step (c) is a step of culturing the plurality of cells in the medium exchanged.
The cells can be cultured in the same manner as described in the above step (a) except that the cells are cultured in a medium not containing extracellular vesicles. The culture time in this step may be a time sufficient for the cells to release extracellular vesicles. For example, it can be 30 minutes or longer, 1 hour or longer, 1.5 hours or longer. Further, for example, it may be 30 minutes to 5 hours, 1 to 4 hours, 1.5 to 3 hours, and the like. For example, the culture time can be 2 hours.
［工程（ｄ）］
　工程（ｄ）は、前記工程（ｃ）後の培地から、細胞外小胞の集団を回収する工程である。
　細胞外小胞は、例えば、工程（ｃ）の後の培養上清から回収することができる。例えば、工程（ｃ）の後、培養液の遠心分離やフィルターろ過等により、細胞と培養上清とを分離することができる。培養上清から細胞外小胞を回収する方法は、特に限定されず、公知の方法を用いることができる。例えば、細胞外小胞の回収方法としては、超遠心分離、限外ろ過、連続フロー電気泳動、クロマトグラフィー等、の方法が挙げられる。
　このようにして、細胞外小胞の集団を得ることができる。 [Step (d)]
Step (d) is a step of collecting a population of extracellular vesicles from the medium after step (c).
Extracellular vesicles can be recovered, for example, from the culture supernatant after step (c). For example, after the step (c), the cells and the culture supernatant can be separated by centrifuging the culture solution or filtering the filter. The method for recovering extracellular vesicles from the culture supernatant is not particularly limited, and a known method can be used. For example, methods for recovering extracellular vesicles include ultracentrifugation, ultrafiltration, continuous flow electrophoresis, chromatography and the like.
In this way, a population of extracellular vesicles can be obtained.
　本実施形態の製造方法により得られる細胞外小胞の集団は、細胞周期を同調させた細胞から放出されたものであり、品質の揃った細胞外小胞の集団である。品質が揃った細胞外小胞の集団とは、例えば、細胞外小胞のゼータ電位の標準偏差が任意の閾値以下である細胞外小胞の集団である。例えば、細胞外小胞のゼータ電位の標準偏差が、５ｍＶ以下、好ましくは４．５ｍＶ以下、より好ましくは４ｍＶ以下、さらに好ましくは３．５ｍＶ以下、特に好ましくは３ｍＶ以下である、細胞外小胞の集団である。 The population of extracellular vesicles obtained by the production method of the present embodiment is a population of extracellular vesicles released from cells synchronized in cell cycle and of uniform quality. The group of extracellular vesicles having uniform quality is, for example, a group of extracellular vesicles in which the standard deviation of the zeta potential of the extracellular vesicles is an arbitrary threshold value or less. For example, the extracellular vesicles whose standard deviation of zeta potential of extracellular vesicles is 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, further preferably 3.5 mV or less, particularly preferably 3 mV or less. It is a group of.
　本実施形態の製造方法により得られる細胞外小胞の集団は、上記のように品質が揃ったものであるため、ＤＤＳのキャリアや再生医療等の医薬品としての利用に適している。 Since the population of extracellular vesicles obtained by the production method of the present embodiment has the same quality as described above, it is suitable for use as a drug for DDS carriers and regenerative medicine.
［任意工程］
　本実施形態の製造方法は、上記工程（ａ）～（ｄ）に加えて、任意の工程を含んでいてもよい。任意の工程は、特に限定されないが、例えば、工程（ｄ）の後、（ｅ）回収した細胞外小胞の集団に含まれる細胞外小胞のゼータ電位を測定する工程等が挙げられる。 [Optional process]
The manufacturing method of this embodiment may include an optional step in addition to the steps (a) to (d). Although an arbitrary process is not specifically limited, For example, (e) The process of measuring the zeta potential of the extracellular vesicle contained in the collect | recovered extracellular vesicle group etc. is mentioned after a process (d).
（工程（ｅ））
　本実施形態の製造方法は、工程（ｄ）により回収した細胞外小胞の集団に含まれる細胞外小胞のゼータ電位を測定する工程を含んでいてもよい。前記細胞外小胞のゼータ電位の測定は、公知の方法で行うことができる。細胞外小胞のゼータ電位の測定方法としては、例えば、上記「＜細胞外小胞の集団＞」で記載した方法等が挙げられる。 (Process (e))
The production method of this embodiment may include a step of measuring the zeta potential of extracellular vesicles contained in the population of extracellular vesicles collected in step (d). The zeta potential of the extracellular vesicle can be measured by a known method. Examples of the method for measuring the zeta potential of extracellular vesicles include the method described in the above “<Extracellular vesicle population>”.
　本実施形態の製造方法は、さらに、（ｆ）工程（ｅ）により得られたゼータ電位の標準偏差を算出する工程を含んでいてもよい。そして、さらに、（ｇ）前記標準偏差が、所定の閾値以下である細胞外小胞の集団を選択する工程を含んでいてもよい。前記標準偏差の閾値としては、例えば５ｍＶ以下、好ましくは４．５ｍＶ以下、より好ましくは４ｍＶ以下、さらに好ましくは３．５ｍＶ以下、特に好ましくは３ｍＶ以下が挙げられる。 The manufacturing method of the present embodiment may further include a step of (f) calculating a standard deviation of the zeta potential obtained by the step (e). Further, (g) a step of selecting a population of extracellular vesicles in which the standard deviation is not more than a predetermined threshold value may be included. Examples of the standard deviation threshold include 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, still more preferably 3.5 mV or less, and particularly preferably 3 mV or less.
　本実施形態の製造方法は、工程（ａ）～（ｄ）により品質の揃った細胞外小胞の集団を得ることができるが、上記工程（ｅ）～（ｇ）を行なうことにより、さらに品質の揃った細胞外小胞の集団を得ることができる。 In the production method of the present embodiment, a population of extracellular vesicles having uniform quality can be obtained by steps (a) to (d). However, the quality can be further improved by carrying out steps (e) to (g). A group of extracellular vesicles with uniform thickness can be obtained.
＜細胞外小胞の集団の品質を評価する方法＞
　１実施形態において、本発明は、細胞外小胞の集団の品質を評価する方法を提供する。本実施形態の方法は、（ａ）細胞外小胞の集団に含まれる複数の細胞外小胞のゼータ電位を測定する工程と、（ｂ）前記工程（ａ）で測定されたゼータ電位の標準偏差を算出する工程と、（ｃ）前記工程（ｂ）で算出された標準偏差に基づいて、前記細胞外小胞の集団の品質を評価する工程、を含む。 <Method for evaluating the quality of a population of extracellular vesicles>
In one embodiment, the present invention provides a method for assessing the quality of a population of extracellular vesicles. The method of this embodiment includes (a) a step of measuring zeta potential of a plurality of extracellular vesicles contained in a population of extracellular vesicles, and (b) a standard of zeta potential measured in the step (a). And (c) evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b).
［工程（ａ）］
　工程（ａ）は、細胞外小胞の集団に含まれる複数の細胞外小胞のゼータ電位を測定する工程である。細胞外小胞の集団中の個々の細胞外小胞のゼータ電位の測定は、公知の方法で行うことができる。細胞外小胞のゼータ電位の測定方法としては、例えば、上記「＜細胞外小胞の集団＞」で記載した方法が挙げられる。 [Step (a)]
Step (a) is a step of measuring the zeta potential of a plurality of extracellular vesicles contained in the population of extracellular vesicles. Measurement of the zeta potential of individual extracellular vesicles in a population of extracellular vesicles can be performed by a known method. Examples of the method for measuring the zeta potential of extracellular vesicles include the method described in the above “<Extracellular vesicle population>”.
［工程（ｂ）］
　工程（ｂ）は、前記工程（ａ）で測定されたゼータ電位の標準偏差を算出する工程である。ゼータ電位の標準偏差の算出は、上記「＜細胞外小胞の集団＞」で記載した方法により行うことができる。 [Step (b)]
Step (b) is a step of calculating the standard deviation of the zeta potential measured in step (a). The standard deviation of the zeta potential can be calculated by the method described above in “<population of extracellular vesicles>”.
［工程（ｃ）］
　工程（ｃ）は、前記工程（ｂ）で算出された標準偏差に基づいて、前記細胞外小胞の集団の品質を評価する工程である。例えば、細胞外小胞が由来する細胞の種類、細胞外小胞の用途等に基づいて、標準偏差の閾値を設定し、工程（ｂ）で算出された標準偏差が前記閾値以下である場合に、細胞外小胞の集団の均一性が高いと評価することができる。すなわち、細胞外小胞の集団の品質が高い（品質が揃っている）と判断することができる。
　標準偏差の閾値としては、例えば、５ｍＶ以下、好ましくは４．５ｍＶ以下、より好ましくは４ｍＶ以下、さらに好ましくは３．５ｍＶ以下、特に好ましくは３ｍＶ以下等が例示される。 [Step (c)]
Step (c) is a step of evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b). For example, when the standard deviation threshold is set based on the type of cell from which the extracellular vesicle is derived, the use of the extracellular vesicle, etc., and the standard deviation calculated in step (b) is equal to or less than the threshold It can be evaluated that the uniformity of the extracellular vesicle population is high. That is, it can be determined that the quality of the population of extracellular vesicles is high (the quality is uniform).
Examples of the standard deviation threshold include 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, still more preferably 3.5 mV or less, and particularly preferably 3 mV or less.
［任意工程］
　本実施形態の方法は、さらに、上記工程（ｃ）により、均一性が高いと評価された細胞外小胞の集団を選択する工程、を含んでいてもよい。あるいは、上記工程（ｃ）により、均一性が低いと評価された細胞外小胞の集団を廃棄する工程、を含んでいてもよい。 [Optional process]
The method of the present embodiment may further include a step of selecting a population of extracellular vesicles evaluated as having high uniformity by the step (c). Alternatively, the method may include a step of discarding a population of extracellular vesicles evaluated as having low uniformity by the step (c).
　本実施形態の方法によれば、細胞外小胞の集団の品質を管理することができ、品質の揃った細胞外小胞の集団を維持することができる。 According to the method of the present embodiment, the quality of the extracellular vesicle population can be managed, and the extracellular vesicle population with uniform quality can be maintained.
＜組成物＞
　１実施形態において、本発明は、複数の細胞外小胞を含む組成物であって、前記組成物に含まれる細胞外小胞のゼータ電位の標準偏差が５ｍＶ以下である、組成物、を提供する。 <Composition>
In one embodiment, the present invention provides a composition comprising a plurality of extracellular vesicles, wherein the standard deviation of the zeta potential of the extracellular vesicles contained in the composition is 5 mV or less. To do.
　本実施形態の組成物に含まれる細胞外小胞のゼータ電位の標準偏差は、５ｍＶ以下である。前記ゼータ電位の標準偏差は、４．５ｍＶ以下であることが好ましく、４ｍＶ以下であることがより好ましく、３．５ｍＶ以下であることが好ましく、３ｍＶ以下であることがより好ましい。 The standard deviation of the zeta potential of the extracellular vesicles contained in the composition of this embodiment is 5 mV or less. The standard deviation of the zeta potential is preferably 4.5 mV or less, more preferably 4 mV or less, preferably 3.5 mV or less, and more preferably 3 mV or less.
　複数の細胞外小胞は、２個以上であればよく、特に限定されないが、例えば、１０３～１０１５個、１０４～１０１２以上、１０５～１０１０個の細胞外小胞であってもよい。
　本実施形態の組成物が含む複数の細胞外小胞は、上記「＜細胞外小胞の集団＞」で説明した上記実施形態の細胞外小胞の集団である。 The plurality of extracellular vesicles may be two or more, and is not particularly limited. For example, the number of extracellular vesicles is 10 3 to 10 15 , 10 4 to 10 12 or more, 10 5 to 10 10 extracellular vesicles. May be.
The plurality of extracellular vesicles contained in the composition of the present embodiment is the population of extracellular vesicles of the above-described embodiment described in the above “<population of extracellular vesicles>”.
　本実施形態の組成物は、複数の細胞外小胞に加えて、任意の成分を含んでいてもよい。任意の成分は、特に限定されないが、例えば、各種緩衝液（生理食塩水、リン酸緩衝液、ＨＥＰＥＳ緩衝液等）、細胞培養液等が挙げられる。 The composition of the present embodiment may contain an arbitrary component in addition to a plurality of extracellular vesicles. Although arbitrary components are not specifically limited, For example, various buffer solutions (physiological saline, a phosphate buffer, a HEPES buffer etc.), a cell culture solution, etc. are mentioned.
　本実施形態の組成物は、医薬組成物、化粧品、又は食品（機能性食品、健康食品等を含む）等であってもよい。本実施形態の組成物が、医薬組成物、化粧品、又は食品等である場合、その用途に応じて後述の各種成分を含んでいてもよい。 The composition of the present embodiment may be a pharmaceutical composition, cosmetics, food (including functional food, health food, etc.) and the like. When the composition of this embodiment is a pharmaceutical composition, cosmetics, food, or the like, various components described later may be included depending on the application.
＜医薬組成物＞
　１実施形態において、本発明は、上記実施形態の細胞外小胞の集団を含む、医薬組成物を提供する。 <Pharmaceutical composition>
In one embodiment, the present invention provides a pharmaceutical composition comprising the population of extracellular vesicles of the above embodiment.
　上記実施形態の細胞外小胞の集団、又は上記実施形態の製造方法で製造された細胞外小胞の集団（以下、まとめて「本細胞外小胞の集団」という場合がある。）は、均一性が高いため、医薬組成物に含有させることができる。上記実施形態の細胞外小胞の集団は、例えば、薬物を内包するキャリア（例えば、ＤＤＳのキャリア）や再生医療等に利用することができる。 The population of extracellular vesicles of the above embodiment, or the population of extracellular vesicles produced by the production method of the above embodiment (hereinafter sometimes collectively referred to as “the population of extracellular vesicles”). Due to its high uniformity, it can be contained in a pharmaceutical composition. The population of extracellular vesicles of the above-described embodiment can be used, for example, for a carrier containing a drug (for example, a carrier of DDS) or regenerative medicine.
　本実施形態の医薬組成物は、本細胞外小胞の集団に加えて、他の成分を含んでいてもよい。例えば、少なくとも１種の薬学的に許容される担体を含み得る。「薬学的に許容される担体」とは、有効成分の生理活性を阻害せず、且つ、その投与対象に対して実質的な毒性を示さない担体を意味する。「実質的な毒性を示さない」とは、その成分が通常使用される投与量において、投与対象に対して毒性を示さないことを意味する。薬学的に許容される担体は、典型的には非活性成分とみなされる、公知のあらゆる薬学的に許容され得る成分を包含する。薬学的に許容される担体は、特に限定されないが、例えば、溶媒、希釈剤、ビヒクル、賦形剤、流動促進剤、結合剤、造粒剤、分散化剤、懸濁化剤、湿潤剤、滑沢剤、崩壊剤、可溶化剤、安定剤、乳化剤、充填剤、保存剤（例えば、酸化防止剤）、キレート剤、矯味矯臭剤、甘味剤、増粘剤、緩衝剤、着色剤等が挙げられる。溶媒としては、例えば、水、生理食塩水、リン酸緩衝液、ＨＥＰＥＳ緩衝液、細胞培養培地、ＤＭＳＯ、ジメチルアセトアミド、エタノール、グリセロール、ミネラルオイル等が挙げられる。薬学的に許容される担体は、１種を単独で用いてもよく、２種以上を併用してもよい。
　この他、医薬分野において常用される成分を特に制限なく使用することができる。本実施形態の医薬組成物は、例えば、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、ｐＨ調整剤、賦形剤、安定剤、抗酸化剤、浸透圧調整剤、防腐剤、着色剤、香料等を含んでいてもよい。これらは、１種を単独で用いてもよく、２種以上を併用してもよい。 The pharmaceutical composition of this embodiment may contain other components in addition to the population of the present extracellular vesicles. For example, it may contain at least one pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the administration subject. By “not exhibiting substantial toxicity” is meant that the ingredient is not toxic to the administered subject at the doses normally used. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredients that are typically considered inactive ingredients. The pharmaceutically acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (for example, antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. Can be mentioned. Examples of the solvent include water, physiological saline, phosphate buffer, HEPES buffer, cell culture medium, DMSO, dimethylacetamide, ethanol, glycerol, mineral oil, and the like. A pharmaceutically acceptable carrier may be used alone or in combination of two or more.
In addition, components commonly used in the pharmaceutical field can be used without particular limitation. The pharmaceutical composition of the present embodiment includes, for example, a solubilizing agent, a suspending agent, an isotonic agent, a buffer, a pH adjusting agent, an excipient, a stabilizer, an antioxidant, an osmotic pressure adjusting agent, and an antiseptic. , Coloring agents, fragrances and the like may be included. These may be used alone or in combination of two or more.
　また、本実施形態の医薬組成物は、有効成分として、薬理作用を有する薬剤（活性成分）を含んでいてもよい。前記薬剤は、特に限定されず、本実施形態の医薬組成物の用途に応じて適宜選択すればよい。前記薬剤としては、例えば、抗がん剤、ビタミン類及びその誘導体類、消炎剤、抗炎症剤、血行促進剤、刺激剤、ホルモン類、刺激緩和剤、鎮痛剤、細胞賦活剤、植物・動物・微生物エキス、鎮痒剤、消炎鎮痛剤、抗真菌剤、抗ヒスタミン剤、催眠鎮静剤、精神安定剤、抗高血圧剤、降圧利尿剤、抗生物質、麻酔剤、抗菌性物質、抗てんかん剤、冠血管拡張剤、生薬、止痒剤、角質軟化剥離剤等が挙げられるが、これらに限定されない。薬剤は、１種を単独で用いてもよく、２種以上を併用してもよい。
　薬剤は、例えば、本細胞外小胞の集団を構成する細胞外小胞に封入されていてもよい。 Moreover, the pharmaceutical composition of this embodiment may contain the chemical | medical agent (active ingredient) which has a pharmacological action as an active ingredient. The said chemical | medical agent is not specifically limited, What is necessary is just to select suitably according to the use of the pharmaceutical composition of this embodiment. Examples of the drug include anticancer agents, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, stimulus relieving agents, analgesics, cell activators, plants and animals.・ Microbial extract, antipruritics, anti-inflammatory analgesics, antifungals, antihistamines, hypnotic sedatives, tranquilizers, antihypertensives, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptics, coronary vasodilation Examples include, but are not limited to, agents, herbal medicines, antipruritic agents, and keratin softening release agents. A medicine may be used individually by 1 type and may use 2 or more types together.
The drug may be encapsulated in, for example, extracellular vesicles that constitute the population of the present extracellular vesicles.
　本実施形態の医薬組成物の剤型は、特に限定されず、医薬品製剤として一般的に用いられる剤型とすることができる。本実施形態の医薬組成物の剤型としては、例えば、錠剤、被覆錠剤、丸剤、散剤、顆粒剤、カプセル剤、液剤、懸濁剤、乳剤等の経口的に投与する剤型、あるいは、注射剤、坐剤、皮膚外用剤等の非経口的に投与する剤型等が挙げられる。これらの剤型の医薬組成物は、定法（例えば、日本薬局方記載の方法）に従って、製剤化することができる。 The dosage form of the pharmaceutical composition of the present embodiment is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation. Examples of the dosage form of the pharmaceutical composition of the present embodiment include orally administered dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, and emulsions, or Examples include dosage forms administered parenterally such as injections, suppositories, and external preparations for skin. The pharmaceutical composition of these dosage forms can be formulated according to a conventional method (for example, a method described in the Japanese Pharmacopoeia).
　本実施形態の医薬組成物の投与経路は、特に限定されず、経口又は非経口経路で投与することができる。なお、非経口経路は、経口以外の全ての投与経路、例えば、静脈内、筋肉内、皮下、鼻腔内、皮内、点眼、脳内、直腸内、腟内及び腹腔内等への投与を包含する。投与は、局所投与であっても全身投与であってもよい。
　本実施形態の医薬組成物は、単回投与又は複数回投与を行うことが可能であり、その投与期間及び間隔は、薬物の種類、疾患の種類及び状態等、投与経路、投与対象の年齢、体重及び性別等によって、適宜選択することができる。本実施形態の医薬組成物を複数回投与する場合、投与間隔は、例えば、１日１～３回、３日毎、１週間毎等とすることができる。
　本実施形態の医薬組成物の投与量は、薬物の種類、疾患の種類及び状態等、投与経路、投与対象の年齢、体重及び性別等によって、適宜選択することができる。本実施形態の医薬組成物の投与量は、医薬組成物に含まれる薬物の治療的有効量とすることができ、例えば、１回につき体重１ｋｇあたり０．０１～１０００ｍｇ程度、０．１～５００ｍｇ程度、０．１～１００ｍｇ程度等とすることができる。 The administration route of the pharmaceutical composition of this embodiment is not particularly limited, and can be administered by oral or parenteral routes. The parenteral route includes all routes other than oral administration such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, ophthalmic, intracerebral, intrarectal, intravaginal, intraperitoneal, etc. To do. Administration may be local or systemic.
The pharmaceutical composition of the present embodiment can be administered in a single dose or multiple doses, and the administration period and interval thereof include the type of drug, the type and condition of the disease, the administration route, the age of the administration subject, It can be appropriately selected depending on body weight and sex. When the pharmaceutical composition of this embodiment is administered a plurality of times, the administration interval can be, for example, 1 to 3 times a day, every 3 days, every week, etc.
The dosage of the pharmaceutical composition of the present embodiment can be appropriately selected depending on the type of drug, the type and condition of the disease, the administration route, the age, weight and sex of the administration subject. The dosage of the pharmaceutical composition of the present embodiment can be a therapeutically effective amount of the drug contained in the pharmaceutical composition, for example, about 0.01 to 1000 mg per kg body weight at a time, 0.1 to 500 mg. About 0.1 to 100 mg.
＜化粧品＞
　１実施形態において、本発明は、上記実施形態の細胞外小胞の集団を含む、化粧品を提供する。 <Cosmetics>
In one embodiment, the present invention provides a cosmetic product comprising the population of extracellular vesicles of the above embodiment.
　本実施形態の化粧品は、本細胞外小胞の集団に加えて、適宜他の成分を含んでいてもよい。本実施形態の化粧品は、化粧品の種類に応じた既知の方法に従って、製造することができる。他の成分としては、特に限定されず、例えば、薬学的に許容される担体等が例示される。薬学的に許容される担体としては、上記「＜医薬組成物＞」で例示したものと同様のものが例示される。本実施形態の化粧品は、化粧品添加物として公知の材料を他の成分として用いてもよい。他の成分は、１種を単独で用いてもよく、２種以上を併用してもよい。 The cosmetic product of this embodiment may contain other components as appropriate in addition to the population of the present extracellular vesicles. The cosmetic of this embodiment can be manufactured according to a known method according to the type of cosmetic. Other components are not particularly limited, and examples thereof include pharmaceutically acceptable carriers. Examples of the pharmaceutically acceptable carrier are the same as those exemplified in the above “<Pharmaceutical composition>”. The cosmetics of this embodiment may use materials known as cosmetic additives as other components. Other components may be used alone or in combination of two or more.
　本実施形態の化粧品は、美容効果等を有する薬剤（活性成分）を含んでいてもよい。前記薬剤は、特に限定されず、本実施形態の化粧品の用途に応じて適宜選択すればよい。前記薬剤としては、例えば、美白材、紫外線吸収剤、育毛用薬剤、収れん剤、抗しわ剤、抗老化剤、ひきしめ剤、制汗剤、保湿剤、ビタミン類及びその誘導体類、消炎剤、抗炎症剤、血行促進剤、刺激剤、ホルモン類、刺激緩和剤、細胞賦活剤、植物・動物・微生物エキス、生薬、止痒剤、角質軟化剥離剤等が挙げられるが、これらに限定されない。薬剤は、１種を単独で用いてもよく、２種以上を併用してもよい。
　薬剤は、例えば、本細胞外小胞の集団を構成する細胞外小胞に封入されていてもよい。 The cosmetic product of this embodiment may contain a drug (active ingredient) having a cosmetic effect or the like. The said chemical | medical agent is not specifically limited, What is necessary is just to select suitably according to the use of the cosmetics of this embodiment. Examples of the agent include whitening materials, ultraviolet absorbers, hair-growth agents, astringents, anti-wrinkle agents, anti-aging agents, tanning agents, antiperspirants, moisturizers, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, Examples include, but are not limited to, inflammatory agents, blood circulation promoters, stimulants, hormones, stimulant mitigators, cell activators, plant / animal / microbe extracts, herbal medicines, antipruritic agents, keratin softening release agents, and the like. A medicine may be used individually by 1 type and may use 2 or more types together.
The drug may be encapsulated in, for example, extracellular vesicles that constitute the population of the present extracellular vesicles.
　本実施形態の化粧品において、化粧品の種類は特に限定されない。化粧品としては、例えば、化粧水、乳液、ローション、クリーム、ジェル、サンスクリーン剤、パック、マスク、美容液などの基礎化粧品；ファンデーション類、化粧下地、口紅類、リップグロス、頬紅類などのメーキャップ化粧品；洗顔剤、ボディーシャンプー、クレンジング剤などの洗浄料；シャンプー、リンス、ヘアコンディショナー、トリートメント、整髪剤などの毛髪用化粧品；ボディーパウダー、ボディーローションなどのボディ用化粧品等が挙げられるが、これらに限定されない。 In the cosmetic product of this embodiment, the type of cosmetic product is not particularly limited. Examples of cosmetics include basic cosmetics such as lotions, emulsions, lotions, creams, gels, sunscreens, packs, masks, and cosmetics; makeup cosmetics such as foundations, makeup bases, lipsticks, lip glosses, and blushers Cleaning agents such as facial cleansers, body shampoos and cleansing agents; hair cosmetics such as shampoos, rinses, hair conditioners, treatments, hair styling agents; and body cosmetics such as body powders and body lotions, but are not limited to these Not.
　本実施形態の化粧品は、化粧品の用途に応じて、通常の化粧品と同様の使用方法で使用することができる。 The cosmetic product of the present embodiment can be used in the same manner as a normal cosmetic product, depending on the use of the cosmetic product.
＜食品＞
　１実施形態において、本発明は、上記実施形態の細胞外小胞の集団を含む、食品を提供する。 <Food>
In one embodiment, the present invention provides a food product comprising the population of extracellular vesicles of the above embodiment.
　本実施形態の食品は、本細胞外小胞の集団に加えて、適宜他の成分を含んでいてもよい。本実施形態の食品は、食品の種類に応じた既知の方法に従って、製造することができる。他の成分としては、特に限定されず、例えば、食品添加物として公知の材料を他の成分として用いてもよい。他の成分は、１種を単独で用いてもよく、２種以上を併用してもよい。 The food of this embodiment may contain other components as appropriate in addition to the population of the present extracellular vesicles. The food of this embodiment can be produced according to a known method according to the type of food. Other components are not particularly limited, and for example, materials known as food additives may be used as other components. Other components may be used alone or in combination of two or more.
　本実施形態の食品において、食品の種類は特に限定されない。食品としては、例えば、そば、うどん、はるさめ、中華麺、即席麺、カップ麺などの各種の麺類；パン、小麦粉、米粉、ホットケーキ、マッシュポテトなどの炭水化物類；青汁、清涼飲料、炭酸飲料、栄養飲料、果実飲料、野菜飲料、乳酸飲料、乳飲料、スポーツ飲料、茶、コーヒーなどの飲料；豆腐、おから、納豆などの豆製品；カレールー、シチュールー、インスタントスープなどの各種スープ類；アイスクリーム、アイスシャーベット、かき氷などの冷菓類；飴、クッキー、キャンディー、ガム、チョコレート、錠菓、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、その他の焼き菓子などの菓子類；かまぼこ、はんぺん、ハム、ソーセージなどの水産・畜産加工食品；加工乳、発酵乳、バター、チーズ、ヨーグルトなどの乳製品；サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシングなどの油脂及び油脂加工食品；ソース、ドレッシング、味噌、醤油、たれなどの調味料；各種レトルト食品、ふりかけ、漬物などのその他加工食品、等を挙げることができるが、これらに限定されない。 In the food of this embodiment, the type of food is not particularly limited. Examples of food include various types of noodles such as buckwheat, udon, harusame, Chinese noodles, instant noodles, cup noodles; carbohydrates such as bread, flour, rice flour, hot cakes, mashed potatoes; green juices, soft drinks, carbonated drinks, Beverages such as nutritional drinks, fruit drinks, vegetable drinks, lactic acid drinks, milk drinks, sports drinks, tea and coffee; bean products such as tofu, okara and natto; various soups such as curry roux, stew roux and instant soup; ice Frozen confectionery such as cream, ice sherbet, shaved ice; sweets such as candy, cookies, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, and other baked confectionery; kamaboko, hampen, ham, sausage Processed fishery and livestock products such as processed milk, fermented milk, butter, cheese, yogurt and other milk Products; salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing and other fats and oils processed foods; sauces, dressings, miso, soy sauce, sauces and other seasonings; various retort foods, sprinkles, pickles Although foodstuff etc. can be mentioned, it is not limited to these.
　本実施形態の食品は、健康食品、機能性食品等であってもよい。この場合、公知の製剤化方法により、乾燥粉末、顆粒剤、錠剤、ゼリー剤、ドリンク剤等に製剤化したものであってもよい。 The food of this embodiment may be health food, functional food, or the like. In this case, it may be formulated into a dry powder, granule, tablet, jelly, drink or the like by a known formulation method.
　本実施形態の食品は、通常の食品と同様に摂取すればよい。 The food of this embodiment may be taken in the same way as normal food.
　本発明を実施例に基づいて説明する。ただし、本発明の実施態様は、これら実施例の記載に限定されるものではない。 The present invention will be described based on examples. However, the embodiment of the present invention is not limited to the description of these examples.
［参考１］
（方法）
　ヒト急性骨髄性白血病細胞であるＨＬ６０細胞を、ウシ胎児血清（ＦＢＳ）を１０％添加したロズウェルパーク記念研究所（ＲＰＭＩ）培地（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）で培養した。細胞外小胞（ＥＶ）回収時には、ＦＢＳ中のＥＶを超遠心法で除去したＥＶ除去培地を用いた。回収した培養上清からのＥＶ精製は以下の手順で行った。まず、細胞片やマイクロベシクル等の大きな粒子を取り除くため、３００×ｇで１０分、２，０００×ｇで２０分、及び１０，０００×ｇで１００分でそれぞれ遠心した。次に、その上清を１００，０００×ｇで２００分遠心し、沈殿物を１０ｍＭのＨＥＰＥＳ緩衝液で懸濁し、ＥＶ試料を得た。ＥＶ試料を液面調節用の流路を並行させた分析チップのマイクロ流路に導入し、散乱光イメージングで個々の粒子の重心位置を可視化し追跡した。それぞれのＥＶに対して、ブラウン運動解析により粒子径を、電気泳動解析によりゼータ電位を見積もった。 [Reference 1]
(Method)
HL60 cells, which are human acute myeloid leukemia cells, were cultured in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS). When collecting extracellular vesicles (EV), an EV removal medium in which EV in FBS was removed by ultracentrifugation was used. EV purification from the collected culture supernatant was performed according to the following procedure. First, to remove large particles such as cell debris and microvesicles, centrifugation was performed at 300 × g for 10 minutes, 2,000 × g for 20 minutes, and 10,000 × g for 100 minutes. Next, the supernatant was centrifuged at 100,000 × g for 200 minutes, and the precipitate was suspended in 10 mM HEPES buffer to obtain an EV sample. The EV sample was introduced into the micro-channel of the analysis chip in which the channel for adjusting the liquid level was arranged in parallel, and the barycentric position of each particle was visualized and tracked by scattered light imaging. For each EV, the particle size was estimated by Brownian motion analysis, and the zeta potential was estimated by electrophoresis analysis.
（結果）
　測定結果の正確性を上げるため、ＨＬ６０細胞の培養上清から精製したＥＶの多点計測を行った（図１７、１８）。ＥＶの粒径とゼータ電位の平均値と標準偏差は、それぞれ１２７±７７ｎｍ、－１２．５±５．４ｍＶであった。ＥＶの粒径は３０ｎｍ付近の粒子まで測定可能であること、ゼータ電位は測定回ごとのバラツキが少なく、信頼性の高い測定が可能であることが示された。 (result)
In order to increase the accuracy of the measurement results, multipoint measurement of EV purified from the culture supernatant of HL60 cells was performed (FIGS. 17 and 18). The average value and standard deviation of the EV particle size and zeta potential were 127 ± 77 nm and −12.5 ± 5.4 mV, respectively. It was shown that the particle size of EV can be measured up to particles near 30 nm, and that the zeta potential has little variation from measurement to measurement, and can be measured with high reliability.
［実施例１］
（方法）
　ヒト急性骨髄性白血病であるＨＬ６０細胞の細胞数を血算板を用いて計数し、初期細胞数を１×１０７個に調整した。細胞培養用の培地として、ウシ胎児血清（ＦＢＳ）を１０％添加したＰＲＭＩ培地（通常培地）を用いた。また、通常培地に、細胞をＧ１期に同調させる薬剤であるＬｅｐｔｏｍｙｃｉｎＢ（ＬＭＢ）を１００ｎＭ添加した培地（ＬＭＢ添加培地）を、細胞周期同調用の培地として用いた。また、超遠心法でＥＶを除去したＦＢＳを１０％添加したＰＲＭＩ培地を、ＥＶを実施的に含まない培地（ＥＶ除去培地）として用いた。
　ＨＬ６０細胞を、通常培地又はＬＭＢ添加培地で２４時間培養後、ＥＶ除去培地に交換し、２時間培養した。その後、細胞及び細胞上清を回収した。
　回収した細胞について、細胞内ＤＮＡ量により細胞周期を調べた。具体的には、細胞をエタノール固定し、２４時間以上４℃で保存した後に、ＰＢＳを用いて洗浄した。その後、ＲＮＡを分解するためにＲＮａｓｅＡを加えて３７℃で１時間インキュベートした後、プロピジウムイオダイド（ＰＩ）を２時間以上反応させてＤＮＡを染色し、フローサイトメトリー（ＢＤ　ＦＡＣＳＡｒｉａ（登録商標）ＩＩＩｕ、ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ）で蛍光強度を測定した。
　また、回収した培養上清から以下の手順でＥＶを精製した。細胞片やマイクロベシクル等の大きな粒子を取り除くため、３００×ｇで１０分、２，０００×ｇで２０分、及び１０，０００×ｇで１００分で、それぞれ遠心した。次に、その上清を１００，０００×ｇで２００分遠心し、沈殿物を１０ｍＭ　ＨＥＰＥＳ緩衝液に懸濁した。調製したＥＶ試料を、液面差補償型分析チップの流路に導入し、高精度単一ナノ粒子測定システム（図１９参照）にセットして粒径、ゼータ電位を計測した（図２０参照）。
　細胞培養からＥＶ回収までの手順の概略を図２１に示す。細胞からのＥＶ精製方法の手順の概略を図２２に示す。 [Example 1]
(Method)
The number of HL60 cells, which are human acute myeloid leukemia, was counted using a blood count plate, and the initial number of cells was adjusted to 1 × 10 7 . As a medium for cell culture, a RPMI medium (normal medium) supplemented with 10% fetal bovine serum (FBS) was used. Further, a medium (LMB-added medium) obtained by adding 100 nM Leptomycin B (LMB), which is an agent for synchronizing cells to the G1 phase, in a normal medium was used as a medium for cell cycle synchronization. In addition, a PRMI medium supplemented with 10% FBS from which EV was removed by ultracentrifugation was used as a medium that does not contain EV (EV removal medium).
HL60 cells were cultured in a normal medium or a medium supplemented with LMB for 24 hours, then replaced with an EV removal medium, and cultured for 2 hours. Thereafter, cells and cell supernatant were collected.
For the collected cells, the cell cycle was examined by the amount of intracellular DNA. Specifically, the cells were fixed with ethanol, stored at 4 ° C. for 24 hours or more, and then washed with PBS. Thereafter, RNase A was added to degrade RNA and incubated at 37 ° C. for 1 hour, followed by reaction with propidium iodide (PI) for 2 hours or longer to stain DNA, and flow cytometry (BD FACSAria (registered trademark) IIIu). , BD Biosciences).
In addition, EV was purified from the collected culture supernatant by the following procedure. To remove large particles such as cell debris and microvesicles, centrifugation was performed at 300 × g for 10 minutes, 2,000 × g for 20 minutes, and 10,000 × g for 100 minutes. Next, the supernatant was centrifuged at 100,000 × g for 200 minutes, and the precipitate was suspended in 10 mM HEPES buffer. The prepared EV sample was introduced into the flow path of the liquid level difference compensation type analysis chip, set in a high-precision single nanoparticle measurement system (see FIG. 19), and the particle size and zeta potential were measured (see FIG. 20). .
An outline of the procedure from cell culture to EV recovery is shown in FIG. An outline of the procedure of the method for purifying EV from cells is shown in FIG.
（結果）
　ＰＩで染色したＤＮＡの蛍光強度をＦＡＣＳで測定した結果を図２３に示す。ＬＭＢ添加培地で培養した細胞は、通常培地（ＬＭＢ無添加培地）で培養した細胞に比べ、Ｓ期の細胞が３６．４％から１４．５％に、Ｇ２／Ｍ期の細胞が２２．２％から１４．５％に減少した。一方、Ｇ１期の細胞が４１．４％から６９．７％に増加した。ＬＭＢにより、Ｇ１期からＳ期への移行が阻害され、細胞周期がＧ１期に同調していることが確認された（図２３）。 (result)
FIG. 23 shows the result of FACS measurement of the fluorescence intensity of DNA stained with PI. The cells cultured in the LMB-added medium are 36.4% to 14.5% in the S phase cells and 22.2 in the G2 / M phase, compared to the cells cultured in the normal medium (without the LMB medium). From 1% to 14.5%. On the other hand, G1 phase cells increased from 41.4% to 69.7%. LMB inhibited the transition from the G1 phase to the S phase, confirming that the cell cycle was synchronized with the G1 phase (FIG. 23).
　ＥＶの粒径とゼータ電位の測定結果を図２４に示す。通常培地で培養した細胞由来のＥＶの粒径並びにゼータ電位の平均値及び標準偏差は、それぞれ、１２９±８０．３ｎｍ、及び－１２．２±５．７３ｍＶであった。一方、ＬＭＢ添加培地で培養した細胞由来のＥＶの粒径並びにゼータ電位の平均値及び標準偏差は、それぞれ、１９３±１１５ｎｍ、及び－１３．４±２．９３ｍＶであった。通常培地で培養した細胞由来のＥＶに比べ、ＬＭＢ添加培地で培養した細胞由来のＥＶでは、ゼータ電位の分布範囲が小さくなった。Ｇ１期に細胞周期を同調させたことで、細胞から分泌されるＥＶのゼータ電位の分布範囲（標準偏差）が小さくなったことから、細胞周期ごとに分泌されるＥＶが異なるということが分かった。
　Ｇ１期に細胞周期を同調させた細胞から分泌されたＥＶでは、ゼータ電位の標準偏差が２．９３ｍＶであり、細胞周期を同調させなかった細胞から分泌されたＥＶの標準偏差（５．７３ｍＶ）よりも、標準偏差の値が小さかった。この結果から、細胞周期を同調させることで、ＥＶのゼータ電位の標準偏差を小さくできることが確認された。 The measurement results of EV particle size and zeta potential are shown in FIG. The average particle size and standard deviation of EV particle size and zeta potential derived from cells cultured in normal medium were 129 ± 80.3 nm and −12.2 ± 5.73 mV, respectively. On the other hand, the average value and standard deviation of the particle size and zeta potential of EVs derived from cells cultured in the LMB-added medium were 193 ± 115 nm and −13.4 ± 2.93 mV, respectively. Compared to EVs derived from cells cultured in a normal medium, EV-derived cells cultured in a medium supplemented with LMB had a smaller zeta potential distribution range. By synchronizing the cell cycle in the G1 phase, the distribution range (standard deviation) of the zeta potential of the EV secreted from the cells was reduced, and it was found that the EV secreted for each cell cycle was different. .
In EV secreted from cells synchronized in the cell cycle in the G1 phase, the standard deviation of the zeta potential is 2.93 mV, and the standard deviation of EV secreted from cells not synchronized in the cell cycle (5.73 mV) The standard deviation value was smaller. From this result, it was confirmed that the standard deviation of the zeta potential of EV can be reduced by synchronizing the cell cycle.
　本発明によれば、品質の揃った細胞外小胞の集団及び当該細胞外小胞を含む組成物等、当該細胞外小胞を製造する方法、並びに細胞外小胞の品質を評価する方法が提供される。本発明の細胞外小胞は、品質が揃っているため、医薬品、化粧品、食品等の様々な用途に利用できる。 According to the present invention, there is provided a method for producing an extracellular vesicle and a method for evaluating the quality of the extracellular vesicle, such as a group of extracellular vesicles having a uniform quality and a composition containing the extracellular vesicle. Provided. Since the extracellular vesicles of the present invention have the same quality, they can be used for various uses such as pharmaceuticals, cosmetics, and foods.
　１　　粒子検出システム
　５　　制御装置
　１００　　粒子検出装置 1 Particle Detection System 5 Control Device 100 Particle Detection Device

Claims (16)

  1. 　細胞外小胞のゼータ電位の標準偏差が５ｍＶ以下である、細胞外小胞の集団。 A group of extracellular vesicles having a standard deviation of zeta potential of the extracellular vesicle of 5 mV or less.
  2. 　前記細胞外小胞がエクソソームである、請求項１に記載の細胞外小胞の集団。 The population of extracellular vesicles according to claim 1, wherein the extracellular vesicles are exosomes.
  3. 　（ａ）複数の細胞の細胞周期を同調させる工程と、
    　（ｂ）前記工程（ａ）後、前記複数の細胞の培地を、細胞外小胞を実質的に含まない培地に交換する工程と、
    　（ｃ）前記培地交換した培地で、前記複数の細胞を培養する工程と、
    　（ｄ）前記工程（ｃ）後の培地から、細胞外小胞の集団を回収する工程と、
    　を含む、細胞外小胞の集団の製造方法。     (A) synchronizing the cell cycle of a plurality of cells;
    (B) after the step (a), replacing the medium of the plurality of cells with a medium substantially free of extracellular vesicles;
    (C) culturing the plurality of cells with the medium exchanged;
    (D) recovering a population of extracellular vesicles from the medium after step (c);
    A method for producing a population of extracellular vesicles, comprising:
  4. 　前記工程（ｄ）後に得られる細胞外小胞の集団が、請求項１又は２に記載の細胞外小胞の集団である、請求項３に記載の細胞外小胞の集団の製造方法。 The method for producing an extracellular vesicle population according to claim 3, wherein the extracellular vesicle population obtained after the step (d) is the extracellular vesicle population according to claim 1 or 2.
  5. 　前記工程（ａ）を、細胞周期同調剤を含む培地で、前記複数の細胞を培養することにより行う、請求項３又は４に記載の細胞外小胞の集団の製造方法。 The method for producing a population of extracellular vesicles according to claim 3 or 4, wherein the step (a) is performed by culturing the plurality of cells in a medium containing a cell cycle synchronizer.
  6. 　前記工程（ａ）を、コンフルエントな状態で、前記複数の細胞を培養することにより行う、請求項３又は４に記載の細胞外小胞の集団の製造方法。 The method for producing an extracellular vesicle population according to claim 3 or 4, wherein the step (a) is performed by culturing the plurality of cells in a confluent state.
  7. 　前記工程（ａ）において、前記複数の細胞をＧ１期に同調させる、請求項３～６のいずれか一項に記載の細胞外小胞の集団の製造方法。 The method for producing a population of extracellular vesicles according to any one of claims 3 to 6, wherein in the step (a), the plurality of cells are synchronized with the G1 phase.
  8. 　前記工程（ｄ）の後、さらに、（ｅ）前記の回収した細胞外小胞の集団に含まれる細胞外小胞のゼータ電位を測定する工程を含む、請求項３～７のいずれか一項に記載の細胞外小胞の集団の製造方法。 The step (d) further comprises the step of (e) measuring the zeta potential of the extracellular vesicles contained in the collected extracellular vesicle population. A method for producing a population of extracellular vesicles as described in 1. above.
  9. 　請求項３～８のいずれか一項に記載の細胞外小胞の集団の製造方法により製造された、細胞外小胞の集団。 A population of extracellular vesicles produced by the method for producing a population of extracellular vesicles according to any one of claims 3 to 8.
  10. 　細胞外小胞の集団の品質を評価する方法であって、
    　（ａ）細胞外小胞の集団に含まれる複数の細胞外小胞のゼータ電位を測定する工程と、
    　（ｂ）前記工程（ａ）で測定されたゼータ電位の標準偏差を算出する工程と、
    　（ｃ）前記工程（ｂ）で算出された標準偏差に基づいて、前記細胞外小胞の集団の品質を評価する工程と、
    　を含む、方法。     A method for assessing the quality of a population of extracellular vesicles comprising:
    (A) measuring the zeta potential of a plurality of extracellular vesicles contained in a population of extracellular vesicles;
    (B) calculating a standard deviation of the zeta potential measured in the step (a);
    (C) evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b);
    Including a method.
  11. 　前記工程（ｃ）において、前記標準偏差が５ｍＶ以下である場合に、前記細胞外小胞の集団の均一性が高いと判定する、請求項１０に記載の細胞外小胞の集団の品質を評価する方法。 The quality of the population of extracellular vesicles according to claim 10, wherein in the step (c), the uniformity of the population of extracellular vesicles is judged to be high when the standard deviation is 5 mV or less. how to.
  12. 　請求項１又は２に記載の細胞外小胞の集団を含む、組成物。 A composition comprising the population of extracellular vesicles according to claim 1 or 2.
  13. 　請求項１又は２に記載の細胞外小胞の集団を含む、医薬組成物。 A pharmaceutical composition comprising the population of extracellular vesicles according to claim 1 or 2.
  14. 　請求項１又は２に記載の細胞外小胞の集団を含む、化粧品。 Cosmetics comprising the population of extracellular vesicles according to claim 1 or 2.
  15. 　請求項１又は２に記載の細胞外小胞の集団を含む、食品。 A food comprising the population of extracellular vesicles according to claim 1 or 2.
  16. 　健康食品又は機能性食品である、請求項１５に記載の食品 The food according to claim 15, which is a health food or a functional food.
PCT/JP2019/008235 2018-03-02 2019-03-01 Extracellular-vesicle population and method for manufacturing same WO2019168184A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2020503662A JP7296937B2 (en) 2018-03-02 2019-03-01 Method for producing extracellular vesicle population

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862637397P 2018-03-02 2018-03-02
US62/637,397 2018-03-02

Publications (1)

Publication Number Publication Date
WO2019168184A1 true WO2019168184A1 (en) 2019-09-06

Family

ID=67805439

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/008235 WO2019168184A1 (en) 2018-03-02 2019-03-01 Extracellular-vesicle population and method for manufacturing same

Country Status (2)

Country Link
JP (1) JP7296937B2 (en)
WO (1) WO2019168184A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7371975B1 (en) 2022-09-27 2023-10-31 セルソース株式会社 Method for producing extracellular vesicles and composition containing extracellular vesicles
JP7423016B1 (en) 2022-09-27 2024-01-29 セルソース株式会社 Extracellular vesicle secretion medium

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7372294B2 (en) 2021-09-27 2023-10-31 矢崎総業株式会社 terminal insertion device

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008174508A (en) * 2007-01-19 2008-07-31 National Institute Of Advanced Industrial & Technology New piericidine derivative or its salt, method for producing the same and antitumor agent containing the derivative as active ingredient
JP2013540150A (en) * 2010-10-18 2013-10-31 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ Use of exosomes to promote or enhance hair growth
WO2014030590A1 (en) * 2012-08-24 2014-02-27 国立大学法人東京大学 Exosome analysis method, exosome analysis apparatus, antibody-exosome complex, and exosome electrophoresis chip
KR20170103697A (en) * 2016-03-03 2017-09-13 고려대학교 산학협력단 Composition for preventing or treating of hepatic fibrosis comprising exosome or exosomal RNAs
JP2017534629A (en) * 2014-11-07 2017-11-24 株式会社エキソステムテックＥｘｏｓｔｅｍｔｅｃｈ Ｃｏ．，Ｌｔｄ． Composition for induction of adipocyte differentiation, regeneration of adipose tissue, skin whitening or wrinkle improvement containing exosomes derived from stem cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008174508A (en) * 2007-01-19 2008-07-31 National Institute Of Advanced Industrial & Technology New piericidine derivative or its salt, method for producing the same and antitumor agent containing the derivative as active ingredient
JP2013540150A (en) * 2010-10-18 2013-10-31 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ Use of exosomes to promote or enhance hair growth
WO2014030590A1 (en) * 2012-08-24 2014-02-27 国立大学法人東京大学 Exosome analysis method, exosome analysis apparatus, antibody-exosome complex, and exosome electrophoresis chip
JP2017534629A (en) * 2014-11-07 2017-11-24 株式会社エキソステムテックＥｘｏｓｔｅｍｔｅｃｈ Ｃｏ．，Ｌｔｄ． Composition for induction of adipocyte differentiation, regeneration of adipose tissue, skin whitening or wrinkle improvement containing exosomes derived from stem cells
KR20170103697A (en) * 2016-03-03 2017-09-13 고려대학교 산학협력단 Composition for preventing or treating of hepatic fibrosis comprising exosome or exosomal RNAs

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7371975B1 (en) 2022-09-27 2023-10-31 セルソース株式会社 Method for producing extracellular vesicles and composition containing extracellular vesicles
JP7423016B1 (en) 2022-09-27 2024-01-29 セルソース株式会社 Extracellular vesicle secretion medium

Also Published As

Publication number Publication date
JPWO2019168184A1 (en) 2021-02-12
JP7296937B2 (en) 2023-06-23

Similar Documents

Publication Publication Date Title
Huda et al. Potential use of exosomes as diagnostic biomarkers and in targeted drug delivery: progress in clinical and preclinical applications
WO2019168184A1 (en) Extracellular-vesicle population and method for manufacturing same
Zhang et al. Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging
Dolati et al. Nanocurcumin improves regulatory T-cell frequency and function in patients with multiple sclerosis
US20200150131A1 (en) Diagnostic advanced glycation end-product antibodies
Fathallah et al. The effect of small oligomeric protein aggregates on the immunogenicity of intravenous and subcutaneous administered antibodies
Barnich et al. CEACAM6 acts as a receptor for adherent-invasive E. coli, supporting ileal mucosa colonization in Crohn disease
EP2889623B1 (en) Exosome analysis method, exosome analysis apparatus, and use of exosome electrophoresis chip
Curran et al. GM-CSF production by glioblastoma cells has a functional role in eosinophil survival, activation, and growth factor production for enhanced tumor cell proliferation
Kong et al. Extracellular acidosis modulates the endocytosis and maturation of macrophages
CN103958546A (en) Modulation of macrophage activation
CN106955353B (en) Acyl-coenzyme A: application of cholesterol acyltransferase ACAT 1inhibitor
US20200055048A1 (en) Optofluidic device and method for detecting circulating tumour cells
Kraus et al. Evaluation of a 3D human artificial lymph node as test model for the assessment of immunogenicity of protein aggregates
CN102427817A (en) Therapeutic pharmaceutical agent for diseases associated with decrease in function of GNE protein, food composition, and food additive
Horgan et al. Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling
Group Young Researchers in Inflammatory Carcinogenesis et al. The heterogeneity of the tumor microenvironment as essential determinant of development, progression and therapy response of pancreatic cancer
KR101564325B1 (en) Composition for prevention or treatment of inflamatory disease comprising culture medium of adipose-derived stem cell-T cell
JP2014110785A (en) One cell screening method for cells secreting and/or displaying physiologically active substance and immunochamber used for the same
US11221287B2 (en) Methods of measuring structural and functional changes of a biomolecular composition
Skovronova et al. Adipose stromal cells bioproducts as cell-free therapies: manufacturing and therapeutic dose determine in vitro functionality
Nakajima et al. Subvisible particles derived by dropping stress enhance anti-PEG antibody production and clearance of PEGylated proteins in mice
Gabrijel et al. Quantification of cell hybridoma yields with confocal microscopy and flow cytometry
Juhl Methodologic and genetic influence on immunohistochemical demonstration and semiquantitation of blood group antigen A in human ureter urothelium.
RU2788905C2 (en) Diagnostic antibodies to advanced glycation end products

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19759905

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020503662

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19759905

Country of ref document: EP

Kind code of ref document: A1